Production of fucosyllactose in a host cell

Abstract

The present invention belongs to the technical fields of synthetic biology and metabolic engineering. More specifically, the present invention belongs to the technical field of fermenting metabolically engineered host cells. The present invention describes a method for producing fucosyllactose by fermenting genetically modified cells, and the genetically modified cells used in this method. The genetically modified cells comprise at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis. More specifically, the cells comprise a nucleic acid sequence encoding a fucosyltransferase for synthesizing fucosyllactose, and comprise at least one nucleic acid expressing a membrane protein, more specifically a nucleic acid sequence expressing a membrane protein capable of transporting fucosyllactose.

Description

Field of the Invention

[0001] The present invention belongs to the technical fields of synthetic biology and metabolic engineering. More specifically, the present invention belongs to the technical field of fermenting metabolically engineered host cells. The present invention describes a method for producing fucosyllactose by fermenting genetically modified cells, as well as the genetically modified cells used in this method. The genetically modified cells comprise at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis. More specifically, the cells comprise a nucleic acid sequence encoding a fucosyltransferase for synthesizing fucosyllactose, and comprise at least one nucleic acid expressing a membrane protein, more specifically a nucleic acid sequence expressing a membrane protein capable of transporting fucosyllactose. Background of the Invention

[0003] Today, more than 80 compounds belonging to the human milk oligosaccharide (HMO) family have been structurally characterized. These HMOs represent a class of complex oligosaccharides with prebiotic functions. In addition, the structural homology of HMOs with epithelial epitopes explains their protective properties against bacterial pathogens. In the infant gastrointestinal tract, HMOs selectively nourish the growth of selected bacterial strains and thus promote the development of a unique gut microbiota in breastfed infants.

[0004] Some of these human milk oligosaccharides require specific fucosylated structures that are most likely to exhibit specific biological activities. The production of these fucosylated oligosaccharides requires the action of fucosyltransferases. These fucosyltransferases belong to the enzyme family of glycosyltransferases and are widely expressed in vertebrates, invertebrates, plants, fungi, yeasts and bacteria. They catalyze the transfer of fucose residues from a donor (usually guanosine diphosphate fucose (GDP-fucose)) to receptors, including oligosaccharides, (glyco)proteins and (glyco)lipids. The thus fucosylated receptor substrates are involved in a variety of biological and pathological processes.

[0005] In the microbial fermentative production of fucosyllactose (FL), FL is in many cases produced intracellularly in industrial production hosts. A problem that has been identified as truly difficult in the field for producing oligosaccharides in cells is the intracellular enrichment of the produced oligosaccharides and their extraction. Intracellular enrichment is thought to be responsible for product inhibition of the production of the desired oligosaccharides. The synthesis may slow down, or the desired oligosaccharides may reach cytotoxic concentrations, leading to metabolic arrest or even cell lysis.

[0006] One object of the present invention is to provide tools and methods by which fucosyllactose can be produced in an efficient, time- and cost-effective manner and a large amount of the desired product can be produced.

[0007] According to the present invention, this and other objects are achieved by providing a method and a cell for producing fucosyllactose, wherein the cell is genetically modified for the production of fucosyllactose and comprises at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis. More specifically, the cell comprises a nucleic acid sequence encoding a fucosyltransferase, thereby synthesizing fucosyllactose. According to the present invention, the cell further expresses a membrane protein. More specifically, the cell further expresses a previously unknown membrane protein to enable fucosyllactose transport. Summary of the Invention Summary of the Invention

[0009] Surprisingly, it has now been found that the membrane protein used in the present invention provides a newly identified membrane protein. More specifically, the present invention provides a newly identified previously unknown membrane protein that is capable of fucosyllactose transport and has a positive effect on the fermentative production of fucosyllactose, providing better yields, productivities, specific productivities, and / or growth rates when used to genetically modify host cells for the production of fucosyllactose.

[0010] The present invention also provides a method for producing fucosyllactose. Fucosyllactose is obtained using a host cell comprising the membrane protein of the present invention.

[0011] Definition

[0012] The words used in this specification to describe the present invention and its various embodiments should be understood not only in their ordinary defined meanings, but also to include, by special definition in this specification, structures, materials, or behaviors that extend beyond the scope of the general definition. Thus, if an element can be understood to have more than one meaning in the context of this specification, its use in the claims must be understood to be general to all possible meanings supported by the specification and the word itself.

[0013] The various embodiments and aspects of the embodiments of the present invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but also to include any order and any combination thereof. When the context requires, all words used in the singular form shall be regarded as including the plural form, and vice versa. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

[0014] In the accompanying drawings and the specification, embodiments of the present invention have been disclosed, and although specific terms are used, these terms are used only in a descriptive sense and not for purposes of limitation. The scope of the present invention is set forth in the following claims. It must be understood that the embodiments shown are set forth only for purposes of illustration and should not be regarded as limiting the present invention. It will be apparent to those skilled in the art that modifications, other embodiments, improvements, details, and uses can be made in accordance with the letter and spirit of what is disclosed herein and within the scope of the present disclosure. The scope of the present disclosure is limited only to the claims as interpreted under patent law, including the doctrine of equivalents. In the following claims, the reference characters used to designate claim steps are provided only for convenience of description and are not intended to imply any particular order of performing the steps.

[0015] As used herein, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to: single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions or single-stranded, double-stranded, and triple-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or triple-stranded regions or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in these regions may be from the same molecule or from different molecules. The regions may include all of one or more molecules, but more typically include regions of only some of the molecules. One of the molecules of the triple-helical region is usually an oligonucleotide. As used herein, the term "polynucleotide" also includes DNA or RNA containing one or more modified bases as described above. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as described herein. In addition, DNA or RNA containing uncommon bases (e.g., inosine) or modified bases (e.g., tritylated bases) should be understood to be encompassed by the term "polynucleotide". It should be understood that a variety of modifications have been made to DNA and RNA for many useful purposes known to those skilled in the art. The term "polynucleotide" as used herein includes such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of characteristic DNA and RNA of viruses and cells (including, for example, simple and complex cells). The term "polynucleotide" also includes short polynucleotides commonly referred to as oligonucleotides.

[0016] "Polypeptide" means any peptide or protein that comprises two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to short chains commonly referred to as peptides, oligopeptides, and oligomers and to longer chains commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids. "Polypeptide" includes those that are modified by natural processes such as processing and other post-translational modifications, as well as by chemical modification techniques. Such modifications are well described in basic textbooks and more detailed monographs, as well as in a vast research literature, and they are well known to those of skill in the art. The same type of modification may be present in several sites of a given polypeptide, in the same or varying degrees. In addition, a given polypeptide may contain multiple types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, transfer-RNA-mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination. Polypeptides may be branched or cyclic, with or without branches. Cyclic, branched, and branched cyclic polypeptides may result from natural processes post-translationally, or may be made by entirely synthetic methods.

[0017] "Isolated" means "altered 'by the hand of man'" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that occurs naturally in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is used herein. Similarly, a "synthetic" sequence, as the term is used herein, means any sequence that has been synthesized and is not directly isolated from a natural source. "Synthetic," as the term is used herein, means any synthesized sequence and is not directly isolated from a natural source.

[0018] "Recombinant" means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host genome and is replicated.

[0019] In the context of the present disclosure, the term "endogenous" refers to any polynucleotide, polypeptide, or protein sequence that is a natural part of a cell and occurs at its natural location in the cell's chromosome.

[0020] The term "heterologous" when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme means that the polynucleotide, gene, nucleic acid, polypeptide, or enzyme is from or derived from a source other than the host biological species. In contrast, "homologous" polynucleotides, genes, nucleic acids, polypeptides, or enzymes are used herein to denote polynucleotides, genes, nucleic acids, polypeptides, or enzymes that are derived from the host biological species. When referring to gene regulatory sequences or auxiliary nucleic acid sequences used to maintain or manipulate gene sequences (e.g., promoters, 5' untranslated regions, 3' untranslated regions, poly A addition sequences, intron sequences, splice sites, ribosome binding sites, internal ribosome entry sequences, genomic homology regions, recombination sites, etc.), "heterologous" means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter that is operably linked to a gene that is not operably linked to it in its natural state (i.e., in the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter", even if the promoter may be from the same species (or in some cases, the same organism) as the gene to which it is linked.

[0021] As used herein, the term "polynucleotide encoding a polypeptide" includes polynucleotides that contain a sequence encoding a polypeptide of the present invention. The term also includes polynucleotides that include a single continuous region or discontinuous regions encoding a polypeptide (e.g., interrupted by an integrated phage or insertion sequence or editing) and may also include additional regions that may contain coding and / or non-coding sequences.

[0022] The term "modified expression" of a gene refers to a change in expression compared to the wild - type expression of said gene at any stage of the fucosyllactose production process. Compared to the wild - type, the modified expression is a lower or higher expression, where the term "higher expression" is also defined as "overexpression" of the gene in the case of an endogenous gene or "expression" in the case of a heterologous gene not present in the wild - type strain. Lower expression is achieved by well - known techniques commonly used by those skilled in the art (such as using siRNA, CrispR, CrispRi, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant genes, knockout genes, transposon mutagenesis...) to alter the gene such that it is less capable (i.e., statistically significantly "less capable" compared to a functional wild - type gene) or completely unable (e.g., knockout - gene) to produce a functional end - product. Overexpression or expression is obtained by well - known techniques commonly used by those skilled in the art, where the gene is part of an "expression cassette" which refers to any sequence in which there is a promoter sequence, untranslated region sequence (containing a ribosome - binding sequence or Kozak sequence), coding sequence (e.g., a membrane protein gene sequence) and optionally a transcription terminator, and which results in the expression of a functionally active protein. The expression is constitutive or conditional or regulated.

[0023] The term "constitutive expression" is defined as expression that is not regulated by transcription factors other than RNA polymerase subunits (such as bacterial σ factors) under specific growth conditions. Non - limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in Escherichia coli (E.coli), or Aft2p, Crz1p, Skn7 in Saccharomyces cerevisiae, or DeoR, GntR, Fur in Bacillus subtilis (B.subtilis). These transcription factors bind to specific sequences and may block or enhance expression under certain growth conditions. RNA polymerase binds to specific sequences to initiate transcription, for example, initiated by a σ factor in a prokaryotic host.

[0024] The term "regulated expression" is defined as expression that is regulated by transcription factors other than RNA polymerase subunits (such as bacterial σ factors) under specific growth conditions. Examples of such transcription factors were described above. Expression regulation is typically achieved by an inducer, such as but not limited to IPTG, arabinose, rhamnose, fucose, allolactose or pH change, or temperature change or carbon consumption or substrate or product produced.

[0025] The term "wild - type" refers to the well - known genetic or phenotypic situation that exists in nature.

[0026] When the term "variant" is used herein, it is a polynucleotide or polypeptide that is respectively different from a reference polynucleotide or polypeptide but retains the basic characteristics. A typical variant of a polynucleotide differs from another reference polynucleotide in its nucleotide sequence. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As described below, nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Generally, the differences are limited, so the sequences of the reference polypeptide and the variant are overall very similar and are identical in many regions. The differences between the variant and the reference polypeptide in the amino acid sequence may consist of one or more substitutions, additions, deletions in any combination. The amino acid residues that are substituted or inserted may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides can be naturally occurring, such as allelic variants, or it can be a variant that is not known to occur in nature. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0027] In some embodiments, the present disclosure contemplates preparing functional variants by modifying the structure of the membrane proteins used in the present invention. Variants can be generated by amino acid substitution, deletion, addition, or a combination thereof. For example, it can be reasonably expected that the replacement of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine alone, or similar replacements of amino acids with structurally related amino acids (e.g., conservative mutations) will not have a significant impact on the biological activity of the resulting molecule. Conservative substitutions are substitutions that occur within amino acid families that are related by side chain. By evaluating the ability of the variant polypeptide to generate a response in cells in a manner similar to the wild-type polypeptide, providing a better yield, productivity, and / or growth rate in the context of the present invention than cells without the variant, it can be easily determined whether a change in the amino acid sequence of the polypeptide of the present disclosure results in a functional homolog.

[0028] As used herein, the term "functional homolog" describes those molecules that have sequence similarity and also share at least one functional characteristic (such as biochemical activity). More specifically, the term "functional homolog" as used herein describes those proteins that have sequence similarity (in other words, homology) and at the same time have at least one functional similarity (such as biochemical activity) (Altenhoff et al., PLoS Comput. Biol. 8 (2012) e1002514). Functional homologs are sometimes referred to as orthologs, where "orthologs" refer to such homologous genes or proteins that are functional equivalents of a reference gene or protein in another species. Functional homologs generally produce the same characteristics to a similar but not necessarily identical extent. Functionally homologous proteins have the same characteristics, where the quantitative measurement produced by one homolog is at least 10% of that of the other; more typically, at least 20%, about 30% to about 40%; for example, about 50% to about 60%; about 70% to about 80%; or about 90% to about 95%; about 98% to about 100%, or greater than 100% of the quantitative measurement produced by the original molecule. Thus, in the case where the molecule has enzyme activity, the functional homolog will have the enzyme activity in the percentages listed above compared to the original enzyme. When the molecule is a DNA-binding molecule (e.g., a polypeptide), the homolog will have the binding affinity in the above percentages, as measured by the weight of the binding molecule compared to the original molecule.

[0029] Functional homologs and reference polypeptides can be naturally occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events.

[0030] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, querying a database of nucleotide or polypeptide sequences can identify homologs of the biomass regulatory polypeptide. Sequence analysis can include BLAST, Reciprocal BLAST, or PSI-BLAST analysis of a non-redundant database using the amino acid sequence of the biomass regulatory polypeptide as a reference sequence. In some cases, the amino acid sequence is deduced from the nucleotide sequence. Generally, those polypeptides in the database with greater than 40% sequence identity are candidates for further evaluation of their suitability as biomass regulatory polypeptides. Amino acid sequence similarity allows for conservative amino acid substitutions, such as one hydrophobic residue being replaced by another or one polar residue being replaced by another. If desired, these candidates can be manually inspected to narrow the number of candidates to be further evaluated. Manual inspection can be carried out by selecting those candidates that appear to have domains (such as conserved functional domains) present in the productivity regulatory polypeptide.

[0031] In the context of polynucleotides, a "fragment" refers to a clone or any portion of a polynucleotide molecule, particularly a polynucleotide portion that retains useful functional characteristics. Useful fragments include oligonucleotides and polynucleotides that can be used in hybridization or amplification techniques or for regulating replication, transcription, or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide, typically having at least about 9 contiguous nucleotides, such as at least about 30 nucleotides or at least about 50 nucleotides of any sequence provided herein. Exemplary fragments may additionally or alternatively include fragments that contain a region encompassing a conserved family domain of a polypeptide, fragments that consist essentially of such a region, or fragments that consist of such a region. Exemplary fragments may additionally or alternatively include fragments that contain a conserved domain of a polypeptide.

[0032] Fragments may additionally or alternatively include subsequences of polypeptide and protein molecules, or subsequences of polypeptides. In some cases, a fragment or domain is a subsequence of a polypeptide that performs at least one biological function of the full-length polypeptide in substantially the same manner or to a similar extent. For example, a polypeptide fragment may contain a recognizable structural motif or functional domain, such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interaction, and may initiate transcription. The size of a fragment can vary from as few as 3 amino acid residues to the full length of the full-length polypeptide, such as at least about 20 amino acid residues in length, such as at least about 30 amino acid residues in length. Preferably, a fragment is a functional fragment that has at least one property or activity of the polypeptide from which it is derived. For example, a fragment may include a functional domain or a conserved domain of a polypeptide. For example, a domain can be characterized by Pfam (https: / / pfam.xfam.org / ) (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432) or the Conserved Domain Database (CDD) (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268). The Pfam database used herein refers to the Pfam database Pfam 32.0 released in September 2018, and the CDD database used herein refers to the CDD database v3.17 released on April 3, 2019.

[0033] The terms "fucosyllactose" ("fucosyllactose", "fucosyllactose") and "FL" used in the present invention are used interchangeably and refer to oligosaccharides containing a fucose residue and a lactose residue. Such fucosyllactose refers to 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose or any combination thereof; fucosyllactose refers to a combination of at least any two of 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose.

[0034] The terms "alpha-1,2'-fucosyltransferase", "alpha 1,2' fucosyltransferase", "2'-fucosyltransferase", "α-1,2'-fucosyltransferase", "α1,2' fucosyltransferase", "2' fucosyltransferase", "2'-FT" or "2'FT" used in the present invention are used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate GDP-L-fucose to a receptor molecule lactose in an α-1,2-bond. A polynucleotide encoding "α-1,2-fucosyltransferase" or any of the above terms refers to a polynucleotide encoding such a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate GDP-L-fucose to a receptor molecule lactose in an α-1,2-bond.

[0035] The terms "2'-fucosyllactose", "alpha-1,2'-fucosyllactose", "alpha 1,2' fucosyllactose", "α-1,2'-fucosyllactose", "α1,2' fucosyllactose", "Fucα1-2Galβ1-4Glc", "2'FL" or "2'-FL" used in the present invention are used interchangeably. In a preferred embodiment, these terms refer to the product obtained by the catalytic transfer of a fucose residue from GDP-L-fucose to lactose in an α-1,2'-bond by α-1,2'-fucosyltransferase.

[0036] The terms "alpha-1,3-fucosyltransferase", "alpha 1,3-fucosyltransferase", "3-fucosyltransferase", "α-1,3-fucosyltransferase", "α1,3-fucosyltransferase", "3 fucosyltransferase", "3-FT" or "3FT" used in the present invention are used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate GDP-L-fucose to a receptor molecule lactose in an α-1,3-bond. A polynucleotide encoding "α-1,3-fucosyltransferase" or any of the above terms refers to a polynucleotide encoding such a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate GDP-L-fucose to a receptor molecule lactose in an α-1,3-bond.

[0037] The terms "3-fucosyllactose", "alpha-1,3-fucosyllactose", "alpha1,3-fucosyllactose", "α-1,3-fucosyllactose", "α1,3-fucosyllactose", "Galβ1-4(Fucα1-3)Glc", "3FL" or "3-FL" used in the present invention are used interchangeably. In a preferred embodiment, these terms refer to the product obtained by transferring a fucose residue from GDP-L-fucose to lactose in an α-1,3-linkage under the catalysis of an α-1,3-fucosyltransferase.

[0038] The terms "difucosyllactose", "di-fucosyllactose", "lactodifucotetraose", "2',3-difucosyllactose", "2',3 difucosyllactose", "α-2',3-fucosyllactose", "α2',3-fucosyllactose", "Fucα1-2Galβ1-4(Fucα1-3)Glc", "DFLac", "2',3-diFL", "DFL" or "diFL" used in the present invention are used interchangeably. In a preferred embodiment, these terms refer to the product obtained by transferring a fucose residue to 2'FL under the catalysis of an α-1,3-fucosyltransferase to obtain 2',3-difucosyllactose, or refer to the product obtained by transferring a fucose residue to 3FL under the catalysis of an α-1,2-fucosyltransferase to obtain 2',3 difucosyllactose.

[0039] The term "oligosaccharide" as used herein and commonly understood in the prior art refers to a sugar polymer containing a small number, typically three to ten, of simple sugars (i.e., monosaccharides).

[0040] As used herein, "SET" or "sugar efflux transporter" refers to membrane proteins of the SET family, which are proteins having the InterPRO domain IPR001214 defined by InterPro 75.0 (release date July 4, 2019) and / or proteins belonging to the eggNOGv4.5 family ENOG410XTE9 defined by the Eggnogdb 1.0.2 database (release date November 3, 2017). Identification of the InterPro domain can be performed using the online tool at https: / / www.ebi.ac.uk / interpro / or a stand-alone version of InterProScan (https: / / www.ebi.ac.uk / interpro / download.html) using default values. Identification of orthologous families in eggNOGv4.5 can be performed using the online version or a stand-alone version of eggNOG-mapperv1 (http: / / eggnogdb.embl.de / # / app / home).

[0041] As used herein, the term "membrane protein" refers to a protein that is part of or interacts with the cell membrane and controls the flow of molecules and information across the cell. Thus, membrane proteins are involved in transport, either into or out of the cell.

[0042] These membrane proteins can be carriers, P-P bond hydrolysis-driven transporters, β-barrel porins, secondary transporters, putative transporters, or phosphotransferase-driven group translocation proteins, as defined by the Transporter Classification Database, operated and maintained by the Bioinformatics Group of the Saier Laboratory (available at www.tcdb.org), which provides functional and phylogenetic classifications of membrane transporters. This Transporter Classification Database details the IUBMB-approved comprehensive classification system for membrane transporters, known as the Transporter Classification (TC) system. The TCDB classification search described herein is based on the definitions of TCDB.org released on June 17, 2019.

[0043] Carriers are a collective term for uniporters, symporters, and antiporters that utilize carrier-mediated processes (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). They belong to the electrochemically potential-driven transporters, also known as secondary carrier-type facilitators. Included in this class are membrane proteins that, when a single species is transported by facilitated diffusion or in a membrane potential-dependent process (if the solute is charged), utilize a carrier-mediated process to catalyze uniport; when two or more species are transported in opposite directions in a tightly coupled process, without coupling to a direct form of energy other than chemiosmotic energy, catalyze antiport; and / or when two or more species are transported together in the same direction in a tightly coupled process, without coupling to a direct form of energy other than the chemiosmotic energy of the secondary carrier, catalyze symport (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188). These systems are generally stereospecific. Solute:solute antiport is a characteristic of secondary carriers. The dynamic association of carriers and enzymes results in functional membrane transport metabolites that directly channel the channel substrates, usually obtained from the extracellular compartment, into their cellular metabolism (Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012), 2687-2706). Solutes transported by these carriers include, but are not limited to, cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated glycolytic intermediates, osmolytes, and siderophores.

[0044] If a membrane protein hydrolyzes the diphosphate bond of inorganic pyrophosphate, ATP, or another nucleoside triphosphate to drive the active uptake and / or extrusion of one or more solutes, the membrane protein belongs to the class of P-P bond hydrolysis-driven transporters (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). The membrane protein may or may not be transiently phosphorylated, but the substrate is not phosphorylated. Substrates transported by P-P bond hydrolysis-driven transporters include, but are not limited to, cations, heavy metals, β-glucans, UDP-glucose, lipopolysaccharides, teichoic acids.

[0045] β-barrel porin membrane proteins form transmembrane pores that generally allow the energy-independent passage of solutes across the membrane. The transmembrane portion of these proteins consists solely of β-strands that form a β-barrel (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). These porin-type proteins are present in the outer membranes of Gram-negative bacteria, mitochondria, plastids, and possibly acid-fast Gram-positive bacteria. Solutes transported by these β-barrel porin membrane proteins include, but are not limited to, nucleosides, raffinose, glucose, β-glucosides, oligosaccharides.

[0046] Ancillary transporters are defined as proteins that facilitate the transport across one or more biological membranes but are not themselves directly involved in the transport. These membrane proteins always function in association with one or more established transport systems, such as, but not limited to, outer membrane factors (OMF), polysaccharide (PST) carriers, ATP-binding cassette (ABC)-type transporters. They can provide energy-related functions coupled to transport, play a structural role in complex formation, provide biological or stability functions, or play a role in regulation (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of ancillary transporters include, but are not limited to, the polysaccharide co-polymerase family involved in polysaccharide transport and the membrane fusion protein family involved in bacteriocin and chemical toxin transport.

[0047] Putative transporters include families that are either classified elsewhere when the transport function of their members is recognized or removed from the transporter classification system when the proposed transport function is negated. These families include one or more members that have been suggested to have a transport function, but the evidence for this function is not yet convincing (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). Examples of putative transporters classified in this group under the TCDB system released on June 17, 2019, include, but are not limited to, copper transporters.

[0048] Phosphotransferase-driven group translocation proteins are also known as PEP-dependent phosphoryl group transfer-driven group translocation proteins of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). The reaction product from extracellular sugar is cytoplasmic sugar-phosphate. The enzyme components that catalyze sugar phosphorylation are superimposed on the transport process in a tightly coupled process. The PTS system is involved in many different aspects, including regulation and chemotaxis, biofilm formation, and pathogenesis (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Saier, J. Mol. Microbiol. Biotechnol. 25 (2015) 73-78). The membrane protein family within the phosphotransferase-driven group translocation proteins classified under the TCDB system released on June 17, 2019, includes PTS systems associated with the transport of glucose-glucoside, fructose-mannitol, lactose-N,N'-diacetylchitobiose-β-glucoside, glucitol, galactitol, mannose-fructose-sorbose, and ascorbate.

[0049] Those skilled in the art should understand that for the databases used herein, including Pfam 32.0 (released in September 2018), CDD v3.17 (released on April 3, 2019), eggnoddb 1.0.2 (released on November 3, 2017), InterPro75.0 (released on July 4, 2019), and TCDB (released on June 17, 2019), the content of each database is fixed at each release and cannot be changed. When the content of a specific database changes, that specific database receives a new release version with a new release date. All release versions of each database, their corresponding release dates, and the specific content annotated at these specific release dates are available to and known by those skilled in the art.

[0050] The term "enable transport" refers to introducing the activity of transporting solutes across the cytoplasmic membrane and / or cell wall. The said transport can be enabled by introducing and / or increasing the expression of the transport proteins described in the present invention. The term "enhance transport" refers to increasing the activity of transporting solutes across the cytoplasmic membrane and / or cell wall. The said transport can be enhanced by introducing and / or increasing the expression of the transport proteins described in the present invention. The "expression" of a transport protein is defined as "overexpression" when the gene encoding the transport protein is an endogenous gene, or "expression" when the gene encoding the transport protein is a heterologous gene that does not exist in the wild-type strain.

[0051] Hybridization

[0052] The term "hybridization" as defined herein is the process in which substantially homologous complementary nucleotide sequences anneal to each other. The hybridization process can occur entirely in solution, i.e., both complementary nucleic acids are in solution. The hybridization process can also be carried out with one of the complementary nucleic acids immobilized to a matrix (e.g., magnetic beads, Sepharose beads, or any other resin). In addition, the hybridization process can be carried out with one of the complementary nucleic acids immobilized to a solid support (such as nitrocellulose or nylon membrane) or immobilized to a siliceous glass support (the latter being referred to as a nucleic acid array or microarray or nucleic acid chip) by, for example, photolithographic techniques. To allow hybridization to occur, nucleic acid molecules are typically heat-denatured or chemically denatured to unwind the double-strand into two single strands and / or to remove hairpins or other secondary structures from single-stranded nucleic acids. The term "stringency" refers to the conditions under which hybridization occurs. The stringency of hybridization is affected by conditions such as temperature, salt concentration, ionic strength, and the composition of the hybridization buffer. Typically, low stringency conditions are selected that are approximately 30 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH value. Medium stringency conditions are 20 °C lower than the Tm, and high stringency conditions are 10 °C lower than the Tm. High stringency hybridization conditions are typically used to isolate hybridization sequences that have a high sequence similarity to the target nucleic acid sequence. However, due to the degeneracy of the genetic code, nucleic acids may vary in sequence and still encode substantially the same polypeptide. Thus, medium stringency hybridization conditions may sometimes be required to identify such nucleic acid molecules.

[0053] The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe at a defined ionic strength and pH value. The Tm depends on the solution conditions and the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. The maximum hybridization rate is obtained at approximately 16 °C to 32 °C below the Tm. The presence of monovalent cations in the hybridization solution reduces the electrostatic repulsion between the two nucleic acid strands, thus facilitating hybridization formation; this effect is visible for sodium concentrations up to 0.4 M (for higher concentrations, this effect can be neglected). Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes by 0.6 to 0.7 °C per % formamide, and the addition of 50% formamide allows hybridization to be carried out at 30 to 45 °C, but the hybridization rate will be reduced. Base pair mismatches reduce the hybridization rate and thermal stability of the duplex. On average, for large probes, the Tm is reduced by approximately 1 °C per % base pair mismatch. The Tm can be calculated using the following formula, depending on the type of hybrid:

[0054] 1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138:267-284, 1984):

[0055] Tm = 81.5 °C + 16.6 x (log10 [Na+]a) + 0.41 x (%[G + Cb] - 500 x [Lc] - 1 - 0.61 x (% formamide)

[0056] 2) DNA - RNA or RNA - RNA hybrids:

[0057] Tm = 79.8 °C + 18.5 x (log10 [Na+]a) + 0.58 x (%[G + Cb]) + 11.8 x (%[G + Cb])2 - 820 x [Lc] - 1

[0058] 3) oligo - DNA or oligo - RNAd hybrids:

[0059] For < 20 nucleotides: Tm = 2 / n

[0060] For 20 - 35 nucleotides: Tm = 22 + 1.46 / n

[0061] Where:

[0062] a: Or for other monovalent cations, but accurate only in the range of 0.01 - 0.4 M.

[0063] b: Accurate only for %GC in the range of 30% to 75%.

[0064] c: L = length of the duplex in base pairs,

[0065] d: oligo, oligonucleotide,

[0066] n: Effective length of the primer = 2 x (number of G + C) + (number of A + T).

[0067] Any of a variety of known techniques can be used to control non - specific binding, e.g., blocking the membrane with a protein - containing solution, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treating with ribonuclease (Rnase). For non - homologous probes, a series of hybridizations can be performed by changing one of the following: (i) gradually decreasing the annealing temperature (e.g., from 68 °C to 42 °C) or (ii) gradually decreasing the formamide concentration (e.g., from 50% to 0%). Those skilled in the art know the various parameters that can be changed during hybridization and that will maintain or change the stringent conditions.

[0068] In addition to the hybridization conditions, the specificity of hybridization usually also depends on the function of the post-hybridization wash. To remove the background generated by non-specific hybridization, the sample is washed with a dilute salt solution. Key factors for this wash include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. The wash conditions are typically carried out at or below the hybridization stringency. The signal generated by a positive hybridization is at least twice that of the background signal. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as described above. Higher or lower stringent conditions can also be selected. Those skilled in the art know the various parameters that can be changed during the wash process and that will maintain or alter the stringent conditions.

[0069] For example, typical high-stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridizing at 65 °C in 1x SSC or at 42 °C in 1x SSC and 50% formamide, followed by washing at 65 °C in 0.3x SSC. Examples of medium-stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridizing at 50 °C in 4x SSC or at 40 °C in 6x SSC and 50% formamide, followed by washing at 50 °C in 2x SSC. The length of the hybrid is the expected length of the hybridizing nucleic acids. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences and identifying conserved regions as described herein. 1x SSC is 0.15 M NaCl and 15 mM sodium citrate; the hybridization solution and wash solution may additionally contain 5x Denhardt's reagent, 0.5 - 1.0% SDS, 100 μg / ml denatured salmon sperm DNA fragments, 0.5% sodium pyrophosphate.

[0070] To define the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and updated annually).

[0071] The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence but not to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 15 °C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at equilibrium (at a specified ionic strength, pH, and nucleic acid concentration). Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5x SSC, and 1% SDS, incubated at 42 °C, or 5x SSC, 1% SDS, incubated at 65 °C, washed in 0.2x SSC and 0.1% SDS at 65 °C.

[0072] The term "purified" refers to a material that is substantially or essentially free of components that interfere with the activity of the biomolecule. For cells, carbohydrates, nucleic acids, and polypeptides, the term "purified" refers to a material that is substantially or essentially free of components that are normally found associated with the material in its native state. Generally, the purified sugars, oligosaccharides, proteins, or nucleic acids of the present invention have a purity of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, typically at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as measured by the band intensity on a silver-stained gel or other methods for determining purity. Purity or homogeneity can be indicated by a variety of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample followed by visualization after staining. For some purposes, high resolution is required and HPLC or similar purification methods are used. For oligosaccharides, such as 3-fucosyllactose, purity can be determined using methods such as, but not limited to, thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis, or mass spectrometry.

[0073] In the case of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity" or % "identity" refer to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned by sequence comparison algorithms or by visual inspection measurements to obtain maximum correspondence. For sequence comparison, one sequence is used as a reference sequence, and the test sequence is compared to it. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and subsequence coordinates are specified if necessary, and the sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the percent sequence identity of one or more test sequences relative to the reference sequence according to the specified program parameters. BLAST and PSI-BLAST can be used to determine percent identity (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402). For the purposes of the present invention, percent identity is determined using MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). For proteins, the following default parameters are used: (1) gap cost for existence: 12, for extension: 2; (2) the matrix employed is BLOSUM50.

[0074] The term "control sequence" refers to a sequence that is recognized by the transcriptional and translational systems of a host cell and permits the transcription and translation of a polynucleotide sequence into a polypeptide. Thus, such DNA sequences are necessary for the expression of a coding sequence operably linked in a particular host cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcriptional terminator sequences. For example, control sequences suitable for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers. If the DNA of a presequence or a secretory leader sequence is expressed as a preprotein involved in polypeptide secretion, then the DNA of the presequence or the secretory leader sequence can be operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, then the promoter or enhancer is effectively linked to the coding sequence; or if a ribosome binding site affects the transcription of a sequence, then the ribosome binding site is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. The control sequence can also be controlled by external chemicals, such as but not limited to IPTG, arabinose, lactose, allolactose, rhamnose, or fucose, via an inducible promoter or via a genetic circuit that induces or inhibits the transcription or translation of the polynucleotide into a polypeptide.

[0075] Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in frame. However, an enhancer does not have to be contiguous.

[0076] As used herein, the term "Cell Productivity Index (CPI)" refers to the mass of product produced by a recombinant cell divided by the mass of recombinant cells produced in the culture. Detailed Description of the Invention

[0078] In a first embodiment, the present invention provides a method for producing fucosyllactose by genetically modified cells. The method comprises the steps of:

[0079] - providing a cell capable of producing fucosyllactose, said cell comprising at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis, more specifically said cell comprising at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a guanosine diphosphate fucose (GDP-fucose) donor to a lactose acceptor to synthesize fucosyllactose;

[0080] - The cell further comprises: i) modified expression of an endogenous membrane protein (more specifically, an endogenous membrane protein involved in fucosyllactose transport, even more specifically, an endogenous membrane protein enabling and / or enhancing fucosyllactose transport), and / or ii) expression of a heterologous membrane protein (more specifically, a heterologous membrane protein involved in fucosyllactose transport, even more specifically, a heterologous membrane protein enabling and / or enhancing fucosyllactose transport), and wherein the membrane protein: i) is selected from the group of membrane proteins comprising any one of the PFAM domains found by searching the genomic neighborhoods of the GT10 and GT11 fucosyltransferase families, the GT10 and GT11 fucosyltransferase families having interpro numbers IPR001503 and IPR002516 as defined in InterPro 75.0 released on July 4, 2019, wherein the genomic neighborhood window size is 14 genes before and 14 genes after each fucosyltransferase, and wherein the membrane protein does not belong to the SET family, or ii) is selected from the group consisting of: membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or functional homologs or functional fragments of any one of the membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or sequences having at least 80% sequence identity to any one of the membrane proteins having SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218; and culturing the cell in a medium under conditions allowing the production of the desired fucosyllactose. As explained herein, fucosyllactose is preferably isolated from the culture.

[0081] In a preferred embodiment of the invention, the host cell comprises a membrane protein selected from the group consisting of:

[0082] a) carriers;

[0083] b) P-P bond hydrolysis-driven transporters;

[0084] c) β-barrel porins;

[0085] d) secondary transporters;

[0086] e) putative transporters; and

[0087] f) phosphotransferase-driven group translocators.

[0088] Another embodiment provides a method for producing fucosyllactose by a genetically modified cell, the method comprising the steps of:

[0089] - Provide cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis (more specifically, a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose),

[0090] - The cells further comprise: i) a modified expression of an endogenous membrane protein involved in fucosyllactose transport (more specifically, an endogenous membrane protein that enables and / or enhances fucosyllactose transport) and / or ii) an expression of a heterologous membrane protein involved in fucosyllactose transport (more specifically, a heterologous membrane protein that enables and / or enhances fucosyllactose transport), and wherein said membrane protein is selected from the group consisting of:

[0091] a) Carriers excluding SET;

[0092] b) Transporters driven by P-P bond hydrolysis;

[0093] c) β-barrel porins;

[0094] d) Cotransporters;

[0095] e) Putative transporters; and

[0096] f) Phosphotransferase-driven group translocation proteins

[0097] - Culturing the cells in a medium under conditions allowing the production of the desired fucosyllactose. As explained herein, preferably, the produced fucosyllactose is isolated from the culture.

[0098] In the method of the present invention described herein, the membrane protein is an endogenous protein with a modified expression, preferably, the endogenous protein is overexpressed; or the membrane protein is a heterologous protein that can be heterologously expressed by the cell. Then, the heterologously expressed membrane protein is introduced and expressed, preferably, overexpressed. In another embodiment, the endogenous protein can have a modified expression in the cell, and the cell also expresses a heterologous membrane protein.

[0099] The host cells used herein are preferably genetically modified to produce fucosyllactose. In a further preferred embodiment, the cells used herein comprise a recombinant fucosyltransferase capable of modifying lactose or an intermediate into fucosyllactose.

[0100] The host cells used herein are optionally genetically modified to produce fucosyllactose, wherein the host cells are modified to express de novo synthesis of GDP-fucose. The de novo synthesis of GDP-fucose is catalyzed by mannose-6-phosphate isomerase, phosphomannomutase encoding gene, mannose-1-phosphate guanylyltransferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase. Preferably, the host cells are further modified to express one or more genes encoding enzymes for de novo synthesis of GDP-fucose.

[0101] By introducing and / or overexpressing lactose permease, the host cells used herein are optionally genetically modified to import lactose into the cells. The lactose permease is encoded, for example, by the lacY gene or the lac12 gene.

[0102] According to another aspect of the present invention, the polynucleotide encoding the membrane protein is adapted to the codon usage of the corresponding cell or expression system.

[0103] In a preferred aspect of the above embodiments, the porter is selected from the group consisting of TCDB classes 2.A.1.1, 2.A.1.12, 2.A.1.15, 2.A.1.2, 2.A.1.3, 2.A.1.36, 2.A.1.38, 2.A.1.46, 2.A.1.68, 2.A.1.7, 2.A.1.81, 2.A.123, 2.A.2, 2.A.21, 2.A.58, 2.A.6.3, 2.A.66, and 2.A.7.1; the P-P bond hydrolysis-driven transporter is selected from the group consisting of TCDB classes 3.A.1.1, 3.A.1.2, 3.A.1.10, 3.A.1.11, and 3.A.1.5; the β-barrel porin is selected from TCDB classes 1.B.18 and 1.B.3.1; the secondary transporter is selected from TCDB class 8.A.3; the putative transporter is selected from the group consisting of TCDB classes 9.B.14 and 9.B.158; or the phosphotransferase-driven group translocation protein is selected from the group consisting of TCDB classes 4.A.1.1 and 4.A.4.1. The TCDB classes are classified according to the definitions published on TCDB.org on June 17, 2019.

[0104] In another preferred aspect of the above-described embodiments, the carriers are selected from the group consisting of eggnog families 05BZS, 05C0R, 05C2C, 05CT4, 05CXP, 05CZQ, 05D94, 05DXI, 05E5M, 05E5W, 05E8G, 05EAM, 05EDR, 05EGZ, 05F9N, 05JHE, 05PSV, 05W2Y, 05W3H, 05XJ5, 070Q9, 07CWC, 07QF7, 07QNK, 07RBJ, 07RJ1, 07T5E, 07VQ3, 0814C, 088QT, 08H15, 08N8A, 08SC4, 08Z4Q; the P-P bond hydrolysis-driven transporters are selected from the group consisting of eggnog families 05BZ1, 05CJ1, 05DMK, 05DFW, 05EY8, 05HAC, 05MFV, 07FKK, 07R5U, 07V1T, 08IJ9, 08JQ7, 172T7; the β-barrel porins are selected from the group consisting of eggnog families 05DAY, 08KDD; the secondary transporters are selected from the group consisting of eggnog family 07SYR; the putative transporters are selected from the group consisting of eggnog families 05CRE, 05GWF, 06N3A; or the phosphotransferase-driven group translocase proteins are selected from the group consisting of eggnog families 05CI1, 05VI0. The eggnog families are classified according to the definition in eggnog 1.0.2 released on November 3, 2017.

[0105] In another preferred aspect of the above-described embodiments, the carriers are selected from the PFAM lists PF00083, PF00474, PF00873, PF00893, PF01895, PF01943, PF02690, PF03083, PF04193, PF05977, PF07690, PF07690, PF13347, PF13440, PF14667; the P-P bond hydrolysis-driven transporters are selected from the PFAM lists PF00005, PF00532, PF00664, PF01061, PF08352, PF14524, PF13407, PF13416, PF17912; the β-barrel porins are selected from the PFAM lists PF02264, PF02563, PF10531, PF18412; the secondary transporters are selected from the PFAM lists PF13807, PF02706; the putative transporters are selected from the PFAM lists PF01578, PF03932, PF05140, PF11045 and / or the phosphotransferase-driven group translocase proteins are selected from the PFAM lists PF00367, PF00358, PF02378, PF03829. The PFAM lists are classified according to the definition in Pfam 32.0 released in September 2018.

[0106] In addition, in another preferred aspect of the above embodiments, the transporter is selected from the InterPro lists IPR000390, IPR001036, IPR001411, IPR001734, IPR001927, IPR002797, IPR003663, IPR003841, IPR004316, IPR004633, IPR004638, IPR004734, IPR004812, IPR005275, IPR005828, IPR005829, IPR006603, IPR010290, IPR011701, IPR020846, IPR023008, IPR023721, IPR023722, IPR026022, IPR027417, IPR027463, IPR029303, IPR032896, IPR036259, IPR038078, IPR038377, IPR039672; the P-P bond hydrolysis-driven transporter is selected from the InterPro lists IPR000412, IPR001734, IPR001761, IPR003439, IPR003593, IPR005829, IPR005978, IPR005981, IPR006059, IPR006060, IPR006061, IPR008995, IPR011527, IPR011701, IPR013456, IPR013525, IPR013563, IPR015851, IPR015855, IPR017871, IPR019554, IPR020846, IPR025997, IPR026266, IPR027417, IPR028082, IPR029439, IPR033893, IPR036259, IPR036640, IPR038377, IPR039421, IPR040582; the β-barrel porin is selected from the InterPro lists IPR003192, IPR003715, IPR019554, IPR023738, IPR036998, IPR040716; the secondary transporter is selected from the InterPro lists IPR003856, IPR020846, IPR027417, IPR032807, IPR036259;The putative transporter is selected from the InterPro lists IPR002541, IPR003439, IPR003593, IPR004316, IPR005627, IPR006603, IPR007816, IPR017871, IPR020368, IPR020846, IPR023648, IPR027417, IPR036259, IPR036822; or the phosphotransferase-driven group translocation protein is selected from the InterPro lists IPR001127, IPR001996, IPR003352, IPR004716, IPR010974, IPR011055, IPR013013, IPR018113, IPR018454, IPR036665, IPR036878. The InterPro lists are classified according to the definitions in InterPro 75.0 released on July 4, 2019.;

[0107] In a preferred aspect of the present invention, the method as described herein uses a host cell expressing a transporter membrane protein selected from: MdfA from Escherichia coli K12 MG1655 having SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 having SEQ ID NO 06, Blon_2331 from Bifidobacterium longum subsp. Infantis (strain ATCC 15697) having SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. Infantis (strain ATCC 15697) having SEQ ID NO 42, a wzx-like protein from Chitinophaga sp. CF118 having SEQ ID NO 58, Prevotella ruminicola (AR32) having SEQ ID NO 66, Lactococcus raffinolactis (ATCC 43920) having SEQ ID NO 64, or Dyadobacter soli DSM 25329 having SEQ ID NO 62, or a functional homolog or functional fragment of any of the above transporter membrane proteins, or a sequence having at least 80% sequence identity to any of the MdfA, IceT, Blon_2331, Blon_2332 membrane proteins or wzx-like membrane proteins having SEQ ID NOs 02, 06, 40, 42, 58, 66, 64 or 62, respectively.In another preferred aspect, the methods described herein use host cells expressing a P-P bond hydrolysis-driven transporter membrane protein selected from: lmrA from Lactococcus lactis SRCM 103457 having SEQ ID NO 28, LpsE membrane proteins from Sporomusa sphaeroides DSM 2875 having SEQ ID NOs 70 and 74 or from Flavobacterium spartansii having SEQ ID NOs 68 and 72, TolC from Candidatus Planktophila sulfonica having SEQ ID NO 76, MsbA from Pedobacter ginsengisoli having SEQ ID NO 82, from Verrucomicrobia bacterium CG1_02_43_26 having SEQ ID NO 84, malE from Escherichia coli K-12 MG1655 having SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 having SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 having SEQ ID NO 214, xylF from Escherichia coli K-12 MG1655 having SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 having SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a sequence having at least 80% sequence identity to any of the lmrA, LpsE, TolC, MsbA, malE, malK, araF, xylF or ytfQ membrane proteins having SEQ ID NOs 28, 70, 74, 68, 72, 76, 82, 84, 206, 208, 214, 216 or 218, respectively.

[0108] In another preferred aspect, the methods described herein use host cells expressing a β-barrel porin selected from: Wza from Escherichia coli K12 MG1655 having SEQ ID NO 34 or lamB from Escherichia coli K-12 MG1655 having SEQ ID NO 204, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity to the Wza or lamB membrane proteins having SEQ ID NOs 34 or 204, respectively.

[0109] In another preferred aspect, the methods described herein use host cells that express an auxiliary transporter selected from: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) having SEQ ID NO 88, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity to the Wzc membrane protein having SEQ ID NO 88.

[0110] In another alternative preferred aspect, the methods described herein use host cells that express a putative transporter selected from: CutC from Clostridium sp. CAG:1013 having SEQ ID NO 90, from Odoribacter splanchnicus DSM20712 having SEQ ID NO 92, from Mitsuaria sp. PDC51 having SEQ ID NO 94, or from Prevotella intermedia ATCC 25611 (DSM 20706) having SEQ ID NO 96, or a functional homolog or functional fragment of the CutC membrane protein, or a sequence having at least 80% sequence identity to any one of the CutC membrane proteins having SEQ ID NOs 90, 92, 94, or 96, respectively.

[0111] In another alternative preferred aspect, the methods described herein use host cells that express a phosphotransferase-driven group translocation protein selected from: nagE from Escherichia coli K-12 MG1655 having SEQ ID NO 210 or srlB from Escherichia coli K-12 MG1655 having SEQ ID NO 212, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity to the nagE or srlB membrane proteins having SEQ ID NOs 210 or 212, respectively.

[0112] In yet another alternative preferred aspect, the methods described herein use host cells that express transporter membrane proteins selected from: MdfA from Escherichia coli K12 MG1655 having SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 having SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 having SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 having SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 having SEQ ID NO 10, YdhC from Escherichia coli K12 MG1655 having SEQ ID NO 12, YbdA from Escherichia coli K12 MG1655 having SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 having SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 having SEQ ID NO 18, YebQ from Escherichia coli K12 MG1655 having SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 having SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 having SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 MG16P having SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 having SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 having SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 50, CDT2 from Neurospora crassa OR74A having SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 having SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 having SEQ ID NO 58,Wzx from Eubacterium sp. CAG:581 with SEQ ID NO 60, Wzx from Acinetobacter soli (DSM 25329) with SEQ ID NO 62, Wzx from Lactococcus raffinolactis (ATCC 43920) with SEQ ID NO 64, Wzx from Prevotella ruminicola (AR32) with SEQ ID NO 66, NAPO from Brachyspira hampsonii P280 / 1 with SEQ ID NO 86, NAm from Actinobaculum suis (DSM 20639) with SEQ ID NO 98, NAm from Ruminococcus gnavus with SEQ ID NO 100, NAm from Curtobacterium sp. 314Chir4.1 with SEQ ID NO 102, Nap from Niabella drilacis (DSM 25811) with SEQ ID NO 104, Nap from Saccharicrinis fermentans (DSM 9555) with SEQ ID NO 106, mdtD from Citrobacter freundii MGH152 with SEQ ID NO 108, mdtD from Citrobacter werkmanii NBRC 105721 with SEQ ID NO 110, mdtD from Citrobacter amalonaticus with SEQ ID NO 112, mdtD from Klebsiella oxytoca with SEQ ID NO 114, mdtD from Escherichia albertii B156 with SEQ ID NO 116, yegB from Salmonella enterica subsp. Salamae with SEQ ID NO 118, mdtD from Klebsiella pneumoniae 30684 / NJST258_2 with SEQ ID NO 120, Tcr_1_D38215 from Klebsiella pneumoniae with SEQ ID NO 122, mdtD from Pseudocitrobacter faecalis with SEQ ID NO 124Cmr from Yokenella regensburgei (ATCC 43003) with SEQ ID NO 126, MdfA from Cronobacter muytjensii with SEQ ID NO 128, MdfA from Klebsiella oxytoca with SEQ ID NO 130, MFS from Citrobacter koseri with SEQ ID NO 132, MdfA from Escherichia marmotae with SEQ ID NO 134, Cmr from Shigella flexneri with SEQ ID NO 136, MdfA from Salmonella enterica subsp. salamae with SEQ ID NO 138, Cmr from Citrobacter youngae (ATCC 29220) with SEQ ID NO 140, MdfA from Citrobacter freundii with SEQ ID NO 142, MdfA from Enterobacter kobei with SEQ ID NO 144, MdfA from Enterobacter sp. with SEQ ID NO 146, MdfA from Lelliottia sp. WB101 with SEQ ID NO 148, MdfA from Enterobacter ludwigii EcWSU1 with SEQ ID NO 150, thaumatin-like protein from Actinoplanes utahensis with SEQ ID NO 152, thaumatin-like protein from Chitinophagaceae bacterium PMG_246 with SEQ ID NO 154, thaumatin-like protein from Rhizobium sp. PDC82 with SEQ ID NO 156, thaumatin-like protein from Kineococcus rhizosphaerae (DSM 19711) with SEQ ID NO 158, thaumatin-like protein from Morganella morganii IS15 with SEQ ID NO 160, thaumatin-like protein from Geodermatophilus obscurus (strain ATCC 25078) with SEQ ID NO 162thaumatin-like protein from Bradyrhizobium sp. BTAi1 having SEQ ID NO 164, thaumatin-like protein from Bradyrhizobium japonicum USDA 110 having SEQ ID NO 166, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria str. 85-10 having SEQ ID NO 168, thaumatin-like protein from Herbaspirillum aquaticum having SEQ ID NO 170, thaumatin-like protein from Flavobacteria bacterium MS024-2A having SEQ ID NO 172, rnd-like protein from Sinorhizobium medicae WSM419 having SEQ ID NO 182, arabinose efflux protein from Azospirillum brasiliense LMG 04375 having SEQ ID NO 184, or a functional homolog or functional fragment of any of the above transporter membrane proteins,or a protein having an amino acid sequence with at least 80% sequence identity to any one of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, class-sweet or arabinose efflux membrane proteins respectively having SEQ ID NO 02, 06, 04, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 32, 38, 40, 42, 46, 48, 50, 52, 54, 58, 60, 62, 64, 66, 86, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 182 or 184; P-P bond hydrolysis-driven transporters selected from: lmrA from Lactococcus lactis strain SRCM103457 having SEQ ID NO 28, OppF from Escherichia coli strain K12 MG1655 having SEQ ID NO 30, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695) having SEQ ID NO 36, Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 44, LpsE from Flavobacterium spartans having SEQ ID NO 68 or 72, LpsE from Myxococcus fulvus DSM 2875 having SEQ ID NO 70 or 74, TolC from Candidatus Planktophila sulfonica having SEQ ID NO 76, TolC from Butyrivibrio hungatei XBD2006 having SEQ ID NO 78, MsbA from Roseburia intestinalis CAG:13 having SEQ ID NO 80, MsbA from Pedobacter ginsengisoli having SEQ ID NO 82, MsbA from Verrucomicrobia bacterium CG1_02_43_26 having SEQ ID NO 84,Wzm from Rhizobium species Root149 with SEQ ID NO 174, Wzm from Azospirillum brasilense LMG 04375 with SEQ ID NO 176, Wzm from Escherichia coli 113303 with SEQ ID NO 196, Wzt from Rhizobium species Root149 with SEQ ID NO 178, Wzt from Azospirillum brasilense LMG 04375 with SEQ ID NO 180, Wzt from Escherichia coli 113303 with SEQ ID NO 194, Nodj from Bradyrhizobium japonicum USDA110 with SEQ ID NO 188 or 190, malE from Escherichia coli K-12 MG1655 with SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 with SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 with SEQ ID NO 214, xylF from Escherichia coli K-12 MG1655 with SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 with SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt or Nodj membrane proteins having SEQ ID NO 28, 30, 36, 44, 68, 72, 70, 74, 76, 78, 80, 82, 84, 174, 176, 196, 178, 180, 194, 188, 190, 206, 208, 214, 216 or 218 respectively; The putative transporters are selected from: cytochrome C biogenesis protein from Helicobacter pylori with SEQ ID NO 56, CutC from Clostridium species CAG:1013 with SEQ ID NO 90, CutC from Odoribacter splanchnicus DSM 20712 with SEQ ID NO 92, CutC from Songjiangella species PDC51 with SEQ ID NO 94, CutC from Prevotella intermedia ATCC25611 (DSM 20706) with SEQ ID NO 96, ybjM from Escherichia coli K12 MG1655 with SEQ ID NO 190, ybjM from Enterobacteriaceae bacterium ENNIH1 with SEQ ID NO 192,or a functional homolog or functional fragment of a polynucleotide encoding any of the aforementioned putative transporters; or a protein having an amino acid sequence with at least 80% sequence identity to any of the CytC, CutC or ybjM membrane proteins having SEQ ID NO: 56, 90, 92, 94, 96, 190 or 192, respectively.,

[0113] As used herein, a protein having an amino acid sequence with at least 80% sequence identity to any of the membrane proteins involved shall be understood to have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity to the full length of the amino acid sequence of the corresponding membrane protein.

[0114] The amino acid sequence of this membrane protein can be a sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 204, 206, 208, 210, 212, 214, 216 or 218 in the attached sequence listing, or an amino acid sequence having at least 80% sequence identity, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% sequence identity with the full-length amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 204, 206, 208, 210, 212, 214, 216 or 218.

[0115] In another aspect of the present invention, the methods described herein use host cells that express a membrane protein, which is a transporter protein involved in the outer membrane transport of compounds across the cell wall.

[0116] In a further preferred aspect, the method for producing fucosyllactose as described herein further comprises at least one of the following steps:

[0117] i) adding a lactose feed to the culture medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the total reactor volume ranges from 250 mL (milliliters) to 10,000 m 3 (cubic meters), preferably added in a continuous manner, and preferably such that the final volume of the culture medium is no more than three times, preferably no more than twice, more preferably less than 2 times the volume of the culture medium before adding the lactose feed;

[0118] ii) adding a lactose feed to the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days through a feed solution;

[0119] iii) adding a lactose feed to the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days through a feed solution, and wherein the concentration of the lactose feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein preferably the pH value of the solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20 °C and 80 °C;

[0120] The method produces a fucosyllactose concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the culture medium.

[0121] Preferably, lactose feeding is achieved by adding lactose at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration of > 300 mM, starting from the beginning of the cultivation.

[0122] On the other hand, lactose feeding is achieved by adding a certain concentration of lactose to the culture medium such that a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM, is obtained during the production phase of the entire cultivation.

[0123] In another embodiment of the method described herein, the host cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner.

[0124] In another embodiment of the method described herein, a carbon and energy source is also added to the culture medium, preferably sucrose, glucose, fructose, glycerol, maltose, maltodextrin, trehalose, polyols, starch, succinate, malate, pyruvate, lactate, ethanol, citrate and / or lactose, preferably continuously added to the culture medium, preferably together with lactose.

[0125] In a preferred embodiment, a carbon substrate, preferably sucrose, is provided in the culture medium for 3 days or longer, preferably up to 7 days; and / or a carbon substrate is provided in the culture medium in a continuous manner at a rate of at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose per liter of the initial culture volume, such that the final volume of the culture medium is no more than three times, advantageously no more than twice, more advantageously less than twice the volume of the culture medium before cultivation.

[0126] Preferably, when performing the method described herein, a first phase of exponential cell growth is provided by adding a carbon substrate, preferably glucose or sucrose, to the culture medium before adding lactose to the culture medium in the second phase.

[0127] In an alternative preferred embodiment, in the method described herein, lactose has been added together with the carbon substrate during the first phase of exponential growth.

[0128] In another embodiment, the method described herein produces only one fucosyllactose selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose and difucosyllactose.

[0129] In an alternative embodiment, the method described herein produces a mixture of fucosyllactoses.

[0130] This mixture may contain at least two selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose and difucosyllactose.

[0131] In the methods described herein, the genetically modified cells are selected from the group consisting of microbial, plant or animal cells, preferably the microorganism is a bacterium, fungus or yeast, preferably the plant is a rice, cotton, rapeseed, soybean, maize or corn plant, preferably the animal is an insect, fish, bird or non-human mammal, as described herein.

[0132] In a specific exemplary embodiment, the method of the present invention produces fucosyllactose in high yield. The method comprises the step of culturing or fermenting in an aqueous or fermentation medium containing lactose, genetically modified cells (preferably Escherichia coli, more preferably Escherichia coli cells modified by knocking out the genes lacZ, lacY, lacA, glgC, agp, pfkA, pfkB, pgi, arcA, iclR, wcaJ, lon and thyA). Even more preferably, the Escherichia coli lacY gene, the fructokinase gene (frk) from Zymomonas mobilis and the sucrose phosphorylase (SP) from Bifidobacterium adolescentis can additionally be knocked into the genome and constitutively expressed. The constitutive promoter is derived from the promoter library described by De Mey et al. (BMC Biotechnology, 2007). These genetic modifications are also described in WO2016075243 and WO2012007481. In addition, the modified Escherichia coli cells have a recombinant gene encoding a single fucosyltransferase, which can be, in an exemplary embodiment, an α-1,3-fucosyltransferase, capable of modifying lactose to produce 3-fucosyllactose (3-FL). The cell also contains a recombinant gene encoding the expression of any one of the membrane proteins as described herein.

[0133] Another aspect of the invention provides a host cell genetically modified to produce fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis (more specifically a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose), and wherein the cell further comprises: i) a modified expression of an endogenous membrane protein involved in fucosyllactose transport, more specifically enabling and / or enhancing fucosyllactose transport; and / or ii) an expression of a heterologous membrane protein involved in fucosyllactose transport, more specifically enabling and / or enhancing fucosyllactose transport. The membrane protein: i) is selected from the group of membrane proteins comprising any one of the PFAM domains found by searching the genomic neighborhoods of the GT10 and GT11 fucosyltransferase families, the GT10 and GT11 fucosyltransferase families having interpro numbers IPR001503 and IPR002516 as defined in InterPro 75.0 released on July 4, 2019, wherein the genomic neighborhood window size is 14 genes before and 14 genes after the respective fucosyltransferase, and wherein the membrane protein does not belong to the SET family, or ii) is selected from the group consisting of: membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or functional homologs or functional fragments of any one of the membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or sequences having at least 80% sequence identity to any one of the membrane proteins having SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218.

[0134] Alternatively or preferably, the membrane protein is selected from the group consisting of:

[0135] a) carriers excluding SET;

[0136] b) transporters driven by P-P bond hydrolysis;

[0137] c) β-barrel porins;

[0138] d) secondary transporters;

[0139] e) putative transporters; and

[0140] f) phosphotransferase-driven group translocators.

[0141] Alternatively or preferably, the membrane protein is selected from the group consisting of: i) transporter membrane proteins selected from: MdfA from Escherichia coli K12 MG1655 having SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 having SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 having SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 having SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 having SEQ ID NO 10, YdhC from Escherichia coli K12 MG1655 having SEQ ID NO 12, YbdA from Escherichia coli K12 MG1655 having SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 having SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 having SEQ ID NO 18, YebQ from Escherichia coli K12 MG1655 having SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 having SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 having SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 having SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 having SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 having SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 50, CDT2 from Neurospora crassa OR74A having SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 having SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 having SEQ ID NO 58, Wzx from Eubacterium sp. CAG:581 having SEQ ID NO 60,Wzx from Agrobacterium tumefaciens (DSM 25329) with SEQ ID NO 62, Wzx from Lactococcus raffinolactis (ATCC 43920) with SEQ ID NO 64, Wzx from Prevotella ruminicola (AR32) with SEQ ID NO 66, NAPO from Brachyspira hampsonii P280 / 1 with SEQ ID NO 86, NAm from Actinobacillus suis (DSM 20639) with SEQ ID NO 98, NAm from Ruminococcus gnavus with SEQ ID NO 100, NAm from Brevibacterium species 314Chir4.1 with SEQ ID NO 102, Nap from Niabella drilacis (DSM 25811) with SEQ ID NO 104, Nap from Saccharicrinis fermentans (DSM 9555) with SEQ ID NO 106, mdtD from Citrobacter freundii MGH152 with SEQ ID NO 108, mdtD from Citrobacter werkmanii NBRC 105721 with SEQ ID NO 110, mdtD from Citrobacter amalonaticus with SEQ ID NO 112, mdtD from Klebsiella oxytoca with SEQ ID NO 114, mdtD from Escherichia albertii B156 with SEQ ID NO 116, yegB from Salmonella enterica subsp. salamae with SEQ ID NO 118, mdtD from Klebsiella pneumoniae 30684 / NJST258_2 with SEQ ID NO 120, Tcr_1_D38215 from Klebsiella pneumoniae with SEQ ID NO 122, mdtD from Citrobacter farmeri with SEQ ID NO 124, Cmr from Prevotella regensburgensis (ATCC 43003) with SEQ ID NO 126, MdfA from Cronobacter muytjensii with SEQ ID NO 128, MdfA from Klebsiella oxytoca with SEQ ID NO 130, MFS from Citrobacter koseri with SEQ ID NO 132, MdfA from Escherichia marmotae with SEQ ID NO 134, Cmr from Shigella flexneri with SEQ ID NO 136, MdfA from Salmonella enterica subsp. salamae with SEQ ID NO 138, Cmr from Citrobacter youngae (ATCC 29220) with SEQ ID NO 140, MdfA from Citrobacter freundii with SEQ ID NO 142MdfA from Enterobacter kobei with SEQ ID NO 144, MdfA from an Enterobacter species with SEQ ID NO 146, MdfA from Lelliottia species WB101 with SEQ ID NO 148, MdfA from Enterobacter ludwigii EcWSU1 with SEQ ID NO 150, thaumatin-like protein from Actinoplanes utahensis with SEQ ID NO 152, thaumatin-like protein from Chitinophagaceae bacterium PMG_246 with SEQ ID NO 154, thaumatin-like protein from Rhizobium species PDC82 with SEQ ID NO 156, thaumatin-like protein from Kineococcus rhizophilus (DSM 19711) with SEQ ID NO 158, thaumatin-like protein from Morganella morganii IS15 with SEQ ID NO 160, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078) with SEQ ID NO 162, thaumatin-like protein from Bradyrhizobium species BTAi1 with SEQ ID NO 164, thaumatin-like protein from Bradyrhizobium japonicum USDA110 with SEQ ID NO 166, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10 with SEQ ID NO 168, thaumatin-like protein from Aquaspirillum itersonii with SEQ ID NO 170, thaumatin-like protein from Flavobacterium bacterium MS024-2A with SEQ ID NO 172, rnd-like protein from Sinorhizobium meliloti WSM419 with SEQ ID NO 182, arabinose efflux protein from Azospirillum brasilense LMG 04375 with SEQ ID NO 184, or a functional homolog or functional fragment of any of the above transporter membrane proteinsor a protein having an amino acid sequence with at least 80% sequence identity to any one of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, class-sweet or arabinose efflux membrane proteins having SEQ ID NO 02, 06, 04, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 32, 38, 40, 42, 46, 48, 50, 52, 54, 58, 60, 62, 64, 66, 86, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 182 or 184; ii) a P-P bond hydrolysis-driven transporter selected from: lmrA from Lactococcus lactis strain SRCM103457 having SEQ ID NO 28, OppF from Escherichia coli strain K12 MG1655 having SEQ ID NO 30, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695) having SEQ ID NO 36, Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 44, LpsE from Flavobacterium spartans having SEQ ID NO 68 or 72, LpsE from Myxococcus fulvus DSM 2875 having SEQ ID NO 70 or 74, TolC from Candidatus Planktophila sulfonica having SEQ ID NO 76, TolC from Vibrio hungatei XBD2006 having SEQ ID NO 78, MsbA from Roseburia intestinalis CAG:13 having SEQ ID NO 80, MsbA from Pedobacter ginsengisoli having SEQ ID NO 82, MsbA from Verrucomicrobia bacterium CG1_02_43_26 having SEQ ID NO 84, Wzm from Rhizobium sp. Root149 having SEQ ID NO 174,Wzm from Azospirillum brasilense LMG 04375 having SEQ ID NO 176, Wzm from Escherichia coli 113303 having SEQ ID NO 196, Wzt from Rhizobium sp. Root149 having SEQ ID NO 178, Wzt from Azospirillum brasilense LMG 04375 having SEQ ID NO 180, Wzt from Escherichia coli 113303 having SEQ ID NO 194, Nodj from Bradyrhizobium japonicum USDA110 having SEQ ID NO 186 or 188, malE from Escherichia coli K-12 MG1655 having SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 having SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 having SEQ ID NO 214, xylF from Escherichia coli MG K-12 MG1655 having SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 having SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt, Nodj, malE, malK, araF, xylF or ytfQ membrane proteins respectively having SEQ ID NO 28, 30, 36, 44, 68, 72, 70, 74, 76, 78, 80, 82, 84, 174, 176, 196, 178, 180, 194, 188, 190, 206, 208, 214, 216 or 218; iii) a putative transporter selected from: cytochrome c biogenesis protein from Helicobacter pylori having SEQ ID NO 56, CutC from Clostridium sp. CAG:1013 having SEQ ID NO 90, CutC from Odoribacter splanchnicus DSM 20712 having SEQ ID NO 92, CutC from Flavobacterium songnenense PDC51 having SEQ ID NO 94, CutC from Prevotella intermedia ATCC 25611 (DSM 20706) having SEQ ID NO 96, ybjM from Escherichia coli K12 MG1655 having SEQ ID NO 190, ybjM from Enterobacteriaceae bacterium ENNIH1 having SEQ ID NO 192, or a functional homolog or functional fragment of any of the above putative transporters,or a protein having an amino acid sequence with at least 80% sequence identity to any one of the CytC, CutC or ybjM membrane proteins having SEQ ID NO 56, 90, 92, 94, 96, 190 or 192, respectively; iv) a β-barrel porin selected from: Wza from Escherichia coli K12 MG1655 having SEQ ID NO 34 or lamB from Escherichia coli K12 MG1655 having SEQ ID NO 204, or a functional homolog or a functional fragment thereof, or a sequence having at least 80% sequence identity to the Wza or lamB membrane protein having SEQ ID NO 34 or 204; v) an auxiliary transporter selected from: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) having SEQ ID NO 88, or a functional homolog or a functional fragment thereof, or a sequence having at least 80% sequence identity to the Wzc membrane protein having SEQ ID NO 88; vi) a phosphotransferase-driven group translocation protein selected from: nagE from Escherichia coli K12 MG1655 having SEQ ID NO 210 or srlB from Escherichia coli K12 MG1655 having SEQ ID NO 212, or a functional homolog or a functional fragment thereof, or a sequence having at least 80% sequence identity to the nagE or srlB membrane protein having SEQ ID NO 210 or 212, respectively.,

[0142] In another aspect of the present invention, the cells as described herein express a membrane protein which is a transporter involved in the outer membrane transport of a compound across the cell wall.

[0143] In another aspect, there are provided cells stably cultured in a culture medium, wherein the culture medium can be any type of growth medium, including minimal medium, complex medium or growth medium, and the growth medium is rich in certain compounds, such as but not limited to vitamins, trace elements, amino acids.

[0144] Preferably, the cells are transformed to contain at least one nucleic acid sequence encoding a protein selected from the group consisting of a lactose transporter, a fucose transporter, a transporter of a nucleotide-activated sugar.

[0145] In the methods described herein, the cell can be a cell of any organism. As used herein, the term "organism" or "cell" refers to a microorganism selected from the list consisting of bacteria, yeast or fungi, or refers to a plant cell, an animal cell, a mammalian cell, an insect cell, and a protozoan cell. The latter bacteria preferably belong to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus. The latter bacteria belonging to the phylum Proteobacteria preferably belong to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacteria preferably relate to any strain belonging to the species Escherichia coli, such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultured Escherichia coli strains - named Escherichia coli K12 strains - which are very well adapted to the laboratory environment and, unlike wild-type strains, have lost the ability to thrive in the intestine. Well-known examples of Escherichia coli K12 strains are K12 wild-type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111, and AA200. Thus, preferably, the present invention specifically relates to the mutant and / or transformed Escherichia coli strains as described above, wherein the Escherichia coli strain is a K12 strain. More specifically, the present invention relates to the mutant and / or transformed Escherichia coli strains as described above, wherein the K12 strain is Escherichia coli MG1655. The latter bacteria belonging to the phylum Firmicutes preferably belong to the genus Bacillus, preferably from the species Bacillus. The latter yeast preferably belongs to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycota. The latter yeast preferably belongs to the genus Saccharomyces, Pichia, Hansunella, Kluyveromyces, Yarrowia, Eremothecium, Zygosaccharomyces, or Debaromyces. The latter fungi preferably belong to the genus Rhizopus, Dictyostelium, or Aspergillus. "Plant cells" include cells of flowering and non-flowering plants, as well as algal cells, such as Chlamydomonas, Chlorella, etc. Preferably, the plant cells are tobacco, alfalfa, rice, tomato, corn, maize or soybean cells; the mammalian cells are CHO cells or HEK cells; the insect cells are Spodoptera frugiperda cells, and the protozoan cells are Leishmania tarentolae cells.

[0146] In a preferred embodiment, the cell is a cell of a microorganism, wherein more preferably, the microorganism is a bacterium or a yeast. In a more preferred embodiment, the microorganism is a bacterium, and most preferably, it is Escherichia coli. Examples of using such Escherichia coli are described herein.

[0147] In another more preferred embodiment, the bacterium is a yeast. The production of fucosyllactose using yeast and examples applicable to the present invention are described by, for example, Hollands et al. (Metabolic Engineering 52 (2019) 232 - 242).

[0148] Generally preferably, the catabolic pathway of the cell for a selected monosaccharide, disaccharide or oligosaccharide is at least partially inactivated, and the monosaccharide, disaccharide or oligosaccharide is involved in the synthesis of fucosyllactose and / or is required for the synthesis of fucosyllactose.

[0149] In another embodiment, the present invention provides a method for producing fucosyllactose, wherein the cells as described herein are used for culturing in a culture medium under conditions allowing the production of the fucosyllactose. Then, the fucosyllactose is isolated from the culture. As used herein, the conditions allowing production should be understood as conditions related to the physical or chemical parameters for the growth of living cells, including but not limited to temperature, pH, pressure, osmotic pressure, and product / segregate concentration. Preferably, such permissive conditions may include a temperature range of 30 + / - 20 °C and a pH range of 7 + / - 3.

[0150] The cells according to the present invention produce fucosyllactose. Fucosyllactose is selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose.

[0151] Another aspect of the present invention provides the use of a membrane protein selected from the group of membrane proteins as defined herein in the fermentative production of fucosyllactose. Fucosyllactose is selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose.

[0152] In another aspect, the present invention provides the use of the cells as defined herein in a method for producing fucosyllactose.

[0153] In yet another aspect, the present invention provides the use of the cells as defined herein, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose, and / or difucosyllactose.

[0154] Furthermore, the present invention also relates to fucosyllactose obtained by the method according to the present invention, and the use of the polynucleotide, vector, host cell, microorganism or polypeptide as described above for the production of fucosyllactose. Fucosyllactose can be used as a food additive, prebiotic, symbiotic, for supplementing infant food, adult food or feed, or as a therapeutic or pharmaceutically active compound. By the novel method, fucosyllactose can be easily and effectively provided without a complex, time-consuming and costly synthesis process.

[0155] As used herein, the term "isolated" refers to harvesting, collecting or recovering fucosyllactose from a host cell and / or its growth medium, as explained herein.

[0156] Fucosyllactose can be isolated from a culture or aqueous medium of a preparation mixture in a conventional manner. If fucosyllactose remains in the cells producing fucosyllactose, fucosyllactose can be released or extracted from the cells using conventional means, such as using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergents, hydrolysis... to disrupt the cells. The culture medium, reaction mixture and / or cell extract, together and separately referred to as the fucosyllactose-containing mixture or culture, can then be further used for the isolation of fucosyllactose.

[0157] Generally, oligosaccharides (fucosyllactose is an oligosaccharide) are purified by first removing the large components (i.e., first removing cells and cell debris), then removing the smaller components (i.e., proteins, endotoxins and other components from 1000 Da to 1000 kDa), and then desalting the oligosaccharides, by retaining the oligosaccharides with a nanofiltration membrane or electrodialysis in the first step and ion exchange (also known as ion exchange chromatography) in the second step, which consists of a cation exchange resin and an anion exchange resin, where most preferably, cation exchange chromatography is carried out before anion exchange chromatography. These steps do not separate sugars with small differences in degree of polymerization from each other. The separation is carried out, for example, by chromatographic separation.

[0158] This preferably relates to clarifying a mixture containing fucosyllactose to remove suspended particles and contaminants, in particular cells, cell components, insoluble metabolites and debris produced by culturing genetically modified cells and / or performing enzymatic reactions. In this step, the mixture containing fucosyllactose can be clarified in a conventional manner. Preferably, the mixture containing fucosyllactose is clarified by centrifugation, flocculation, decantation and / or filtration. The second step of separating fucosyllactose from the mixture containing fucosyllactose preferably involves removing substantially all proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that may interfere with subsequent separation steps, preferably after it has been clarified. In this step, proteins and related impurities can be removed from the mixture containing fucosyllactose in a conventional manner. Preferably, by ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), especially by chromatography, more especially by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography to remove proteins, salts, by-products, colors and other related impurities from the mixture containing fucosyllactose. Except for size exclusion chromatography, proteins and related impurities are retained by the chromatographic medium or the selected membrane, while fucosyllactose remains in the mixture containing fucosyllactose.

[0159] Contaminating compounds with a molecular weight higher than 1000 Da (Daltons) are removed using an ultrafiltration membrane with a cut-off value higher than 1000 Da to about 1000 kDa. The membrane retains the contaminants and the oligosaccharides enter the filtrate. The typical ultrafiltration principle is well known in the art and is based on tubular modules, hollow fibers, spiral wound or plates; they are used in cross-flow conditions or as dead-end filtration. The membrane composition is well known and available from multiple suppliers and consists of PES (polyethersulfone), polyvinylpyrrolidone, PAN (polyacrylonitrile), PA (polyamide), polyvinylidene fluoride (PVDF), NC (nitrocellulose), ceramic materials or combinations thereof.

[0160] Components smaller than the oligosaccharides, such as monosaccharides, salts, disaccharides, acids, bases, culture medium components are separated by nanofiltration or / and electrodialysis. The cut-off molecular weight of such membranes is between 100 Da and 1000 Da. For oligosaccharides such as 2'-fucosyllactose, the optimal cut-off value is 300 Da to 500 Da, thus minimizing losses in the filtrate. The typical membrane composition is well known and is for example polyamide (PA), TFC, PA-TFC, poly(piperazine amide), PES, cellulose acetate or combinations thereof.

[0161] Fucosyllactose is further isolated from the culture medium and / or cells by evaporation, lyophilization, crystallization, precipitation, and / or drying, spray drying, with or without further purification steps. The further purification steps allow for the preparation of fucosyllactose in combination with other oligosaccharides and / or products, such as but not limited to co-formulations obtained by spray drying, drying, or lyophilization or concentrated in liquid form by evaporation.

[0162] In a further aspect, the present invention also provides for the further purification of fucosyllactose. The further purification of fucosyllactose can be accomplished, for example, by removing any residual DNA, proteins, LPS, endotoxins, or other impurities using (activated) carbon or charcoal, nanofiltration, ultrafiltration, or ion exchange. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization or precipitation of the product. Another purification step is spray drying or lyophilizing fucosyllactose.

[0163] Isolated and preferably also purified fucosyllactose can be used as a supplement in infant formulas and for the treatment of various diseases in newborn infants.

[0164] As shown in the examples herein, when using the membrane proteins defined herein, the methods and cells of the present invention provide at least one of the following surprising advantages:

[0165] Compared to a fucosyllactose production host having the same genetic background but lacking the expression of a heterologous membrane protein or the regulated expression of an endogenous membrane protein,

[0166] - A better fucosyllactose titer (enhanced) (g / L),

[0167] - A better productivity r (g fucosyllactose / L / h),

[0168] - A better cell performance index CPI (g fucosyllactose / gram X),

[0169] - A better specific productivity Qp (g fucosyllactose / g X / h),

[0170] - A better sucrose yield Ys (g fucosyllactose / gram sucrose),

[0171] - A better sucrose uptake / conversion rate Qs (g sucrose / g X / h),

[0172] - A better lactose conversion / consumption rate rs (g lactose / h),

[0173] - Enhanced fucosyllactose secretion, and / or

[0174] - An increased growth rate of the production host.

[0175] In addition, the present invention relates to the following specific embodiments:

[0176] 1. A method for producing fucosyllactose by genetically modified cells, comprising the steps of:

[0177] - providing cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis,

[0178] - said cells further comprising: i) modified expression of an endogenous membrane protein and / or ii) expression of a heterologous membrane protein, and wherein said membrane protein is selected from the group of membrane proteins comprising any one of the PFAM domains found by searching the genomic neighborhood of the GT10 and GT11 fucosyltransferase families, said GT10 and GT11 fucosyltransferase families having interpro numbers IPR001503 and IPR002516 respectively, wherein the genomic neighborhood window size is 14 genes before and 14 genes after the respective fucosyltransferase, and wherein said membrane protein does not belong to the SET family,

[0179] - culturing the cells in a medium under conditions allowing the production of the desired fucosyllactose,

[0180] - preferably isolating fucosyllactose from the culture.

[0181] 2. The method according to embodiment 1, wherein said membrane protein is selected from the group consisting of:

[0182] a) carriers;

[0183] b) P-P bond hydrolysis-driven transporters;

[0184] c) β-barrel porins;

[0185] d) secondary transporters; and

[0186] e) putative transporters.

[0187] 3. A method for producing fucosyllactose by genetically modified cells, comprising the steps of:

[0188] - providing cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis,

[0189] - said cells further comprising: i) modified expression of an endogenous membrane protein and / or ii) expression of a heterologous membrane protein, and wherein said membrane protein is selected from the group consisting of:

[0190] a) carriers, and wherein said membrane protein does not belong to the SET family;

[0191] b) Transporters driven by P-P bond hydrolysis;

[0192] c) β-barrel porins;

[0193] d) Secondary transporters; and

[0194] e) Putative transporters,

[0195] - Culturing the cells in a medium under conditions that allow the production of the desired fucosyllactose,

[0196] - Preferably isolating fucosyllactose from the culture.

[0197] 4. The method according to any one of embodiments 2 or 3, wherein the porter is selected from the group consisting of TCDB classes 2.A.1.1, 2.A.1.12, 2.A.1.15, 2.A.1.2, 2.A.1.3, 2.A.1.36, 2.A.1.38, 2.A.1.46, 2.A.1.68, 2.A.1.7, 2.A.1.81, 2.A.123, 2.A.2, 2.A.21, 2.A.58, 2.A.6.3, 2.A.66 and 2.A.7.1.

[0198] 5. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the group consisting of TCDB classes 3.A.1.1, 3.A.1.10, 3.A.1.11 and 3.A.1.5.

[0199] 6. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from TCDB class 1.B.18.

[0200] 7. The method according to any one of embodiments 2 or 3, wherein the secondary transporter is selected from TCDB class 8.A.3.

[0201] 8. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the group consisting of TCDB classes 9.B.14 and 9.B.158.

[0202] 9. The method according to any one of embodiments 2 or 3, wherein the transporter is selected from the group consisting of eggnog families 05BZS, 05C0R, 05C2C, 05CT4, 05CXP, 05CZQ, 05D94, 05DXI, 05E5M, 05E5W, 05E8G, 05EAM, 05EDR, 05EGZ, 05F9N, 05JHE, 05PSV, 05W2Y, 05W3H, 05XJ5, 070Q9, 07CWC, 07QF7, 07QNK, 07RBJ, 07RJ1, 07T5E, 07VQ3, 0814C, 088QT, 08H15, 08N8A, 08SC4, 08Z4Q.

[0203] 10. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the group consisting of eggnog families 05BZ1, 05CJ1, 05EY8, 05HAC, 05MFV, 07V1T, 08IJ9, 08JQ7.

[0204] 11. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from the group consisting of eggnog family 05DAY.

[0205] 12. The method according to any one of embodiments 2 or 3, wherein the cotransporter is selected from the group consisting of eggnog family 07SYR.

[0206] 13. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the group consisting of eggnog families 05CRE, 05GWF, 06N3A.

[0207] 14. The method according to any one of embodiments 2 or 3, wherein the transporter is selected from the PFAM list PF00083, PF00474, PF00873, PF00893, PF01895, PF01943, PF02690, PF03083, PF04193, PF05977, PF07690, PF07690, PF13347, PF13440, and PF14667.

[0208] 15. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the PFAM list PF00005, PF00664, PF01061, PF08352, PF14524, and PF17912.

[0209] 16. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from the PFAM list PF02563, PF10531, and PF18412.

[0210] 17. The method according to any one of embodiments 2 or 3, wherein the auxiliary transporter is selected from the PFAM lists PF13807 and PF02706.

[0211] 18. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the PFAM lists PF01578, PF03932, PF05140, and PF11045.

[0212] 19. The method according to any one of embodiments 2 or 3, wherein the porter is selected from the InterPro lists IPR000390, IPR001036, IPR001411, IPR001734, IPR001927, IPR002797, IPR003663, IPR003841, IPR004316, IPR004633, IPR004638, IPR004734, IPR004812, IPR005275, IPR005828, IPR005829, IPR006603, IPR010290, IPR011701, IPR020846, IPR023008, IPR023721, IPR023722, IPR026022, IPR027417, IPR027463, IPR029303, IPR032896, IPR036259, IPR038078, IPR038377, IPR039672.

[0213] 20. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the InterPro lists IPR000412, IPR001734, IPR003439, IPR003593, IPR005829, IPR005978, IPR005981, IPR008995, IPR011527, IPR011701, IPR013525, IPR013563, IPR015851, IPR017871, IPR019554, IPR020846, IPR027417, IPR029439, IPR036259, IPR036640, IPR038377, IPR039421, and IPR040582.

[0214] 21. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from the InterPro lists IPR003715, IPR019554, and IPR040716.

[0215] 22. The method according to embodiment 2, wherein the auxiliary transporter is selected from the InterPro lists IPR003856, IPR020846, IPR027417, IPR032807, and IPR036259.

[0216] 23. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the InterPro lists IPR002541, IPR003439, IPR003593, IPR004316, IPR005627, IPR006603, IPR007816, IPR017871, IPR020368, IPR020846, IPR023648, IPR027417, IPR036259, and IPR036822.

[0217] 24. The method according to any one of embodiments 2 or 3, wherein the porter membrane protein is selected from: MdfA from Escherichia coli K12 MG1655, IceT from Escherichia coli K12 MG1655, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), wzx-like proteins from Chitinophaga species CF118, Prevotella ruminicola (AR32), Lactococcus raffinolactis (ATCC 43920), or Agrobacterium tumefaciens DSM 25329, or a functional homolog or functional fragment of any of the above porter membrane proteins, or a sequence having at least 80% sequence identity to any of the MdfA, IceT, Blon_2331, Blon_2332 membrane proteins or wzx-like membrane proteins.

[0218] 25. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transport membrane protein is selected from: lmrA from Lactococcus lactis subsp. lactis bv. Diacetylactis, the LpsE membrane protein from Sporidiobolus salmonicolor DSM 2875, Flavobacterium spartans, TolC from Candidatus Planktophila sulfonica, MsbA from Pedobacter ginsengisoli or Verrucomicrobia bacterium CG1_02_43_26, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transport membrane proteins, or a sequence having at least 80% sequence identity to any of the lmrA, LpsE, TolC, or MsbA membrane proteins.

[0219] 26. A method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from: Wza from Escherichia coli K12 MG1655, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity with the Wza membrane protein.

[0220] 27. A method according to any one of embodiments 2 or 3, wherein the auxiliary transporter is selected from: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099), or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity with the Wzc membrane protein.

[0221] 28. A method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from: CutC from Clostridium species CAG:1013, Odoribacter splanchnicus DSM 20712, Inquilinus limosus species PDC51 or Prevotella intermedia ATCC25611 (DSM 20706), or a functional homolog or functional fragment of any of the CutC membrane proteins, or a sequence having at least 80% sequence identity with any of the CutC membrane proteins.

[0222] 29. The method according to any one of embodiments 2 or 3, wherein the transporter membrane protein is selected from: MdfA from Escherichia coli K12 MG1655, IceT from Escherichia coli K12 MG1655, YnfM from Escherichia coli K12 MG1655, Yhhs from Escherichia coli K12 MG1655, EmrD from Escherichia coli K12 MG1655, YdhC from Escherichia coli K12 MG1655, YbdA from Escherichia coli K12 MG1655, YdeE from Escherichia coli K12 MG1655, MhpT from Escherichia coli K12 MG1655, YebQ from Escherichia coli K12 MG1655, YjhB from Escherichia coli K12 MG1655, Bcr from Escherichia coli K12 MG1655, FucP from Escherichia coli K12 MG1655, WzxE from Escherichia coli K12 MG1655, EmrE from Escherichia coli K12 MG1655, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC15697), Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC15697), Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), CDT2 from Neurospora crassa OR74A, CDT2 from Aspergillus oryzae RIB40, Wzx from Chitinophaga sp. CF118, Wzx from Eubacterium sp. CAG:581, Wzx from Agrobacterium tumefaciens (DSM 25329), Wzx from Lactococcus raffinolactis (ATCC43920), Wzx from Prevotella ruminicola (AR32), NAPO from Brachyspira hampsonii P280 / 1, NAm from Actinobacillus suis (DSM 20639), NAm from Ruminococcus gnavus, NAm from Curtobacterium sp. 314Chir4.1, Nam from Planctomycetes bacterium GWF2_42_9, Nap from Niabella drilacis (DSM 25811), Nap from Saccharicrinis fermentans (DSM 9555), mdtD from Citrobacter freundii MGH152, mdtD from Citrobacter werkmanii NBRC 105721, mdtD from Citrobacter amalonaticus, mdtD from Klebsiella oxytoca, mdtD from Escherichia albertii B156,YegB from Salmonella enterica subsp. salamae, mdtD from Klebsiella pneumoniae 30684 / NJST258_2, Tcr_1_D38215 from Klebsiella pneumoniae, mdtD from Citrobacter freundii, Cmr from Prevotella ruminicola (ATCC43003), MdfA from Cronobacter muytjensii, MdfA from Klebsiella oxytoca, MFS from Citrobacter koseri, MdfA from Escherichia marmotae, Cmr from Shigella flexneri, MdfA from Salmonella enterica subsp. salamae, Cmr from Citrobacter youngae (ATCC 29220), MdfA from Citrobacter freundii, MdfA from Enterobacter kobei, MdfA from Enterobacter sp., MdfA from Lelliottia sp. WB101, MdfA from Enterobacter ludwigii EcWSU1, thaumatin-like protein from Actinoplanes utahensis, thaumatin-like protein from Chitinophagaceae bacterium PMG_246, thaumatin-like protein from Rhizobium sp. PDC82, thaumatin-like protein from Kineococcus rhizophilus (DSM 19711), thaumatin-like protein from Morganella morganii IS15, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078), thaumatin-like protein from Bradyrhizobium sp. BTAi1, thaumatin-like protein from Bradyrhizobium japonicum USDA110, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10, thaumatin-like protein from Aquaspirillum itersonii, thaumatin-like protein from Flavobacterium sp. MS024-2A, rnd-like protein from Sinorhizobium meliloti WSM419, arabinose efflux protein from Azospirillum brasilense LMG 04375, or a functional homolog or functional fragment of any of the above transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, thaumatin-like or arabinose efflux membrane proteins.,

[0223] 30. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from: LmrA from Lactococcus lactis strain SRCM103457, OppF from Escherichia coli strain K12 MG1655, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695), Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), LpsE from Flavobacterium spartans, LpsE from Myxococcus fulvus DSM 2875, TolC from Candidatus Planktophila sulfonica, TolC from Butyrivibrio hungatei XBD2006, MsbA from Roseburia intestinalis CAG:13, MsbA from Pedobacter ginsengisoli, MsbA from Verrucomicrobia bacterium CG1_02_43_26, Wzm from Rhizobium sp. Root149, Wzm from Azospirillum brasilense LMG 04375, Wzm from Escherichia coli 113303, Wzt from Rhizobium sp. Root149, Wzt from Azospirillum brasilense LMG 04375, Wzt from Escherichia coli 113303, Nodj from Bradyrhizobium japonicum USDA110, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt, or Nodj membrane proteins.

[0224] 31. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from: cytochrome C biogenesis protein from Helicobacter pylori, CutC from Fusobacterium sp. CAG:1013, CutC from Odoribacter splanchnicus DSM 20712, CutC from Songia sp. PDC51, CutC from Prevotella intermedia ATCC 25611 (DSM20706), ybjM from Escherichia coli K12 MG1655, ybjM from Enterobacteriaceae bacterium ENNIH1, or a functional homolog or functional fragment of any of the above putative transporters, or a protein having an amino acid sequence with at least 80% sequence identity to any of the CytC, CutC, or ybjM membrane proteins.

[0225] 32. A method for producing fucosyllactose by genetically modified cells, comprising the following steps:

[0226] - Provide cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis,

[0227] - Said cells further comprise: i) modified expression of an endogenous membrane protein and / or ii) expression of a heterologous membrane protein, wherein said membrane protein is selected from the group of membrane proteins consisting of:

[0228] Transport membrane proteins: MdfA from Escherichia coli K12 MG1655, IceT from Escherichia coli K12 MG1655, YnfM from Escherichia coli K12 MG1655, Yhhs from Escherichia coli K12 MG1655, EmrD from Escherichia coli K12 MG1655, YdhC from Escherichia coli K12 MG1655, YbdA from Escherichia coli K12 MG1655, YdeE from Escherichia coli K12 MG1655, MhpT from Escherichia coli K12 MG1655, YebQ from Escherichia coli K12 MG1655, YjhB from Escherichia coli K12 MG1655, Bcr from Escherichia coli K12 MG1655, FucP from Escherichia coli K12 MG1655, WzxE from Escherichia coli K12 MG1655, EmrE from Escherichia coli K12 MG1655, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), CDT2 from Neurospora crassa OR74A, CDT2 from Aspergillus oryzae RIB40, Wzx from Chitinophaga sp. CF118, Wzx from Eubacterium sp. CAG:581, Wzx from Agrobacterium tumefaciens (DSM 25329), Wzx from Lactococcus raffinolactis (ATCC 43920), Wzx from Prevotella ruminicola (AR32), NAPO from Brachyspira hampsonii P280 / 1, NAm from Actinobacillus suis (DSM 20639), NAm from Ruminococcus gnavus, NAm from Brevibacterium sp. 314Chir4.1, NAm from Planctomycetes bacterium GWF2_42_9, Nap from Niabella drilacis (DSM25811), Nap from Saccharicrinis fermentans (DSM9555), mdtD from Citrobacter freundii MGH152, mdtD from Citrobacter werkmanii NBRC 105721, mdtD from Citrobacter amalonaticus, mdtD from Klebsiella oxytoca, mdtD from Escherichia albertii B156, yegB from Salmonella enterica subsp. salamae, mdtD from Klebsiella pneumoniae 30684 / NJST258_2,Tcr_1_D38215 from Klebsiella pneumoniae, mdtD from Citrobacter farmeri, Cmr from Prevotella ruminicola (ATCC 43003), MdfA from Cronobacter muytjensii, MdfA from Klebsiella oxytoca, MFS from Citrobacter koseri, MdfA from Escherichia marmotae, Cmr from Shigella flexneri, MdfA from Salmonella enterica subsp. salamae, Cmr from Citrobacter youngae (ATCC 29220), MdfA from Citrobacter freundii, MdfA from Enterobacter kobei, MdfA from Enterobacter species, MdfA from Lelliottia species WB101, MdfA from Enterobacter ludwigii EcWSU1, thaumatin-like protein from Actinoplanes utahensis, thaumatin-like protein from Chitinophagaceae bacterium PMG_246, thaumatin-like protein from Rhizobium species PDC82, thaumatin-like protein from Planococcus rhizosphaerae (DSM 19711), thaumatin-like protein from Morganella morganii IS15, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078), thaumatin-like protein from Bradyrhizobium sp. BTAi1, thaumatin-like protein from Bradyrhizobium japonicum USDA 110, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10, thaumatin-like protein from Aquaspirillum itersonii, thaumatin-like protein from Flavobacterium bacterium MS024-2A, rnd-like protein from Sinorhizobium meliloti WSM419, arabinose efflux protein from Azospirillum brasilense LMG 04375, or any functional homolog or functional fragment of any of the above transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, thaumatin or arabinose efflux membrane protein; and,

[0229] P-P bond hydrolysis-driven transporters: LmrA from Lactococcus lactis strain SRCM103457, OppF from Escherichia coli strain K12 MG1655, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695), Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), LpsE from Flavobacterium spartans, LpsE from Myxococcus fulvus DSM 2875, TolC from Candidatus Planktophila sulfonica, TolC from Butyrivibrio hungatei XBD2006, MsbA from Roseburia intestinalis CAG:13, MsbA from Pedobacter ginsengisoli, MsbA from Verrucomicrobia bacterium CG1_02_43_26, Wzm from Rhizobium sp. Root149, Wzm from Azospirillum brasilense LMG 04375, Wzm from Escherichia coli 113303, Wzt from Rhizobium sp. Root149, Wzt from Azospirillum brasilense LMG 04375, Wzt from Escherichia coli 113303, Nodj from Bradyrhizobium japonicum USDA110, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt or Nodj membrane proteins; and

[0230] β-barrel porin membrane proteins: Wza from Escherichia coli K12 MG1655, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity to the Wza membrane protein; and

[0231] Secondary transporters: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099), or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity to the Wzc membrane protein; and

[0232] Putative transporters: Cytochrome C biosynthesis protein from Helicobacter pylori, CutC from Fusobacterium species CAG:1013, CutC from Clostridium scindens DSM 20712, CutC from Flavobacterium species PDC51, CutC from Prevotella intermedia ATCC 25611 (DSM 20706), ybjM from Escherichia coli K12 MG1655, ybjM from Enterobacteriaceae bacterium ENNIH1, or a functional homolog or functional fragment of any of the foregoing putative transporters, or a protein having an amino acid sequence with at least 80% sequence identity to any of the CytC, CutC or ybjM membrane proteins.

[0233] 33. The method for producing fucosyllactose according to any one of the foregoing embodiments, the method further comprising at least one of the following steps:

[0234] i) adding a lactose feed to the culture medium, comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per initial reactor volume, preferably added in a continuous manner, and preferably such that the final volume of the culture medium is not more than three times, preferably not more than twice, more preferably less than 2 times the volume of the culture medium before adding the lactose feed;

[0235] ii) adding a lactose feed to the culture medium in a continuous manner over a period of 1 day, 2 days, 3 days, 4 days, 5 days through a feed solution;

[0236] iii) adding a lactose feed to the culture medium in a continuous manner over a period of 1 day, 2 days, 3 days, 4 days, 5 days through a feed solution, and wherein the concentration of the lactose feed solution is 50 g / l, preferably 75 g / l, more preferably 100 g / l, more preferably 125 g / l, more preferably 150 g / l, more preferably 175 g / l, more preferably 200 g / l, more preferably 225 g / l, more preferably 250 g / l, more preferably 275 g / l, more preferably 300 g / l, more preferably 325 g / l, more preferably 350 g / l, more preferably 375 g / l, more preferably 400 g / l, more preferably 450 g / l, more preferably 500 g / l, even more preferably 550 g / l, most preferably 600 g / l; wherein preferably the pH of the solution is set to 3 to 7, and wherein preferably the temperature of the feed solution is maintained at 20°C to 80°C;

[0237] iv) The method produces a fucosyllactose concentration of at least 50 g / l, preferably at least 75 g / l, more preferably at least 90 g / l, more preferably at least 100 g / l, more preferably at least 125 g / l, more preferably at least 150 g / l, more preferably at least 175 g / l, more preferably at least 200 g / l in the final volume of the culture medium.

[0238] 34. The method according to embodiment 33, wherein said lactose feeding is achieved by adding lactose at a concentration of at least 5 mM from the start of the culture, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration of > 300 mM.

[0239] 35. The method according to any one of embodiments 33 or 34, wherein said lactose feeding is achieved by adding a certain concentration of lactose to the culture medium such that a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained during the production phase of the entire culture.

[0240] 36. The method according to any one of embodiments 33, 34 or 35, wherein the host cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner.

[0241] 37. The method according to any one of embodiments 33 to 36, wherein a carbon and energy source is also added, preferably sucrose, glucose, fructose, glycerol, maltose, maltodextrin, trehalose, polyol, starch, succinate, malate, pyruvate, lactate, ethanol, citrate, lactose, preferably continuously added to the culture medium, preferably together with lactose.

[0242] 38. The method according to any one of embodiments 33 to 37, wherein a first stage of exponential cell growth is provided by adding a carbon substrate, preferably glucose or sucrose, to the culture medium before adding lactose to the culture medium in the second stage.

[0243] 39. The method according to any one of embodiments 1 to 38, wherein the method produces a mixture of fucosyllactose.

[0244] 40. The method according to any one of embodiments 1 to 39, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose and / or difucosyllactose.

[0245] 41. A method according to any one of embodiments 1 to 40, wherein the genetically modified cell is selected from the group consisting of microbial, plant or animal cells, preferably the microbe is a bacterium, fungus or yeast, preferably the plant is a rice, cotton, rapeseed, soybean, maize or corn plant, preferably the animal is an insect, fish, bird or non-human mammal.

[0246] 42. A method according to embodiment 41, wherein the cell is an Escherichia coli cell.

[0247] 43. A host cell genetically modified for the production of fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding an enzyme involved in the synthesis of fucosyllactose,

[0248] - the cell further comprises: i) a modified expression of an endogenous membrane protein and / or ii) an expression of a heterologous membrane protein, and wherein the membrane protein is selected from the group of membrane proteins comprising any one PFAM domain found by searching the genomic neighborhood of the GT10 and GT11 fucosyltransferase families, the GT10 and GT11 fucosyltransferase families having interpro numbers IPR001503 and IPR002516 respectively, wherein the genomic neighborhood window size is 14 genes before and 14 genes after each fucosyltransferase, and wherein the membrane protein does not belong to the SET family.

[0249] 44. A host cell according to embodiment 43, wherein the membrane protein is selected from the group consisting of:

[0250] a) a porter;

[0251] b) a P-P bond hydrolysis-driven transporter;

[0252] c) a β-barrel porin;

[0253] d) a cotransporter; and

[0254] e) a putative transporter.

[0255] 45. A host cell genetically modified for the production of fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding an enzyme involved in the synthesis of fucosyllactose,

[0256] - the cell further comprises: i) a modified expression of an endogenous membrane protein and / or ii) an expression of a heterologous membrane protein, and wherein the membrane protein is selected from the following group:

[0257] a) a porter, and wherein the membrane protein does not belong to the SET family;

[0258] b) a P-P bond hydrolysis-driven transporter;

[0259] c) β-barrel porin;

[0260] d) secondary transporter; and

[0261] e) putative transporter.

[0262] 46. The host cell according to any one of embodiments 44 or 45, wherein the membrane protein is selected from the group of membrane proteins as defined in any one of embodiments 4 to 32.

[0263] 47. A host cell genetically modified according to any one of embodiments 44 or 45 for the production of fucosyllactose, wherein the transporter membrane protein is selected from: MdfA from Escherichia coli K12 MG1655, IceT from Escherichia coli K12 MG1655, YnfM from Escherichia coli K12 MG1655, Yhhs from Escherichia coli K12 MG1655, EmrD from Escherichia coli K12 MG1655, YdhC from Escherichia coli K12 MG1655, YbdA from Escherichia coli K12 MG1655, YdeE from Escherichia coli K12 MG1655, MhpT from Escherichia coli K12 MG1655, YebQ from Escherichia coli K12 MG1655, YjhB from Escherichia coli K12 MG1655, Bcr from Escherichia coli K12 MG1655, FucP from Escherichia coli K12 MG1655, WzxE from Escherichia coli K12 MG1655, EmrE from Escherichia coli K12 MG1655, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697), CDT2 from Neurospora crassa OR74A, CDT2 from Aspergillus oryzae RIB40, Wzx from Chitinophaga sp. CF118, Wzx from Eubacterium sp. CAG:581, Wzx from Agrobacterium tumefaciens (DSM 25329), Wzx from Lactococcus raffinolactis (ATCC 43920), Wzx from Prevotella ruminicola (AR32), NAPO from Brachyspira hampsonii P280 / 1, NAm from Actinobacillus suis (DSM 20639), NAm from Ruminococcus gnavus, NAm from Brevibacterium sp. 314Chir4.1, NAm from Planctomycetes bacterium GWF2_42_9, Nap from Niabella drilacis (DSM25811), Nap from Saccharicrinis fermentans (DSM9555), mdtD from Citrobacter freundii MGH152, mdtD from Citrobacter werkmanii NBRC 105721, mdtD from Citrobacter amalonaticus, mdtD from Klebsiella oxytoca, mdtD from Escherichia albertii B156, yegB from Salmonella enterica subsp. salamae,MdtD from Klebsiella pneumoniae 30684 / NJST258_2, Tcr_1_D38215 from Klebsiella pneumoniae, mdtD from Citrobacter farmeri, Cmr from Prevotella ruminicola (ATCC43003), MdfA from Cronobacter muytjensii, MdfA from Klebsiella oxytoca, MFS from Citrobacter koseri, MdfA from Escherichia marmotae, Cmr from Shigella flexneri, MdfA from Salmonella enterica subsp. salamae, Cmr from Citrobacter youngae (ATCC 29220), MdfA from Citrobacter freundii, MdfA from Enterobacter kobei, MdfA from an Enterobacter species, MdfA from a Lelliottia species WB101, MdfA from Enterobacter ludwigii EcWSU1, thaumatin-like protein from Actinoplanes utahensis, thaumatin-like protein from Chitinophagaceae bacterium PMG_246, thaumatin-like protein from a Rhizobium species PDC82, thaumatin-like protein from Kineococcus rhizophilus (DSM 19711), thaumatin-like protein from Morganella morganii IS15, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078), thaumatin-like protein from Bradyrhizobium sp. BTAi1, thaumatin-like protein from Bradyrhizobium japonicum USDA110, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10, thaumatin-like protein from Aquaspirillum itersonii, thaumatin-like protein from a Flavobacterium bacterium MS024-2A, rnd-like protein from Sinorhizobium meliloti WSM419, arabinose efflux protein from Azospirillum brasilense LMG 04375, or a functional homolog or functional fragment of any of the above transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, thaumatin or arabinose efflux membrane proteins.,

[0264] 48. A host cell according to any one of embodiments 44 or 45, wherein the P-P bond hydrolysis-driven transporter is selected from: LmrA from Lactococcus lactis strain SRCM103457, OppF from Escherichia coli strain K12MG1655, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695), Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC15697), LpsE from Flavobacterium spartans, LpsE from Myxococcus fulvus DSM 2875, TolC from Candidatus Planktophila sulfonica, TolC from Butyrivibrio hungatei XBD2006, MsbA from Roseburia intestinalis CAG:13, MsbA from Pedobacter ginsengisoli, MsbA from Verrucomicrobia bacterium CG1_02_43_26, Wzm from Rhizobium sp. Root149, Wzm from Azospirillum brasilense LMG 04375, Wzm from Escherichia coli 113303, Wzt from Rhizobium sp. Root149, Wzt from Azospirillum brasilense LMG04375, Wzt from Escherichia coli 113303, Nodj from Bradyrhizobium japonicum USDA110, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a protein having an amino acid sequence with at least 80% sequence identity to any of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt or Nodj membrane proteins.

[0265] 49. A host cell according to any one of embodiments 44 or 45, wherein the putative transporter is selected from: cytochrome C biogenesis protein from Helicobacter pylori, CutC from Fusobacterium sp. CAG:1013, CutC from Odoribacter splanchnicus DSM 20712, CutC from Songia sp. PDC51, CutC from Prevotella intermedia ATCC25611 (DSM 20706), ybjM from Escherichia coli K12 MG1655, ybjM from Enterobacteriaceae bacterium ENNIH1, or a functional homolog or functional fragment of any of the above putative transporters, or a protein having an amino acid sequence with at least 80% sequence identity to any of the CytC, CutC or ybjM membrane proteins.

[0266] 50. A host cell according to any one of embodiments 44 or 45, wherein the β-barrel porin is selected from: Wza from Escherichia coli K12 MG1655, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity with the Wza membrane protein.

[0267] 51. A host cell according to any one of embodiments 44 or 45, wherein the co-transporter is selected from: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099), or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity with the Wzc membrane protein.

[0268] 52. A cell to be stably cultured in a medium, the cell being adjusted to produce fucosyllactose, the cell being transformed to comprise at least one nucleic acid sequence encoding an enzyme involved in fucosyllactose synthesis, characterized in that the cell further comprises: i) modified expression of an endogenous membrane protein and / or ii) expression of a heterologous membrane protein, and wherein the membrane protein is as defined in any one of embodiments 1 to 33.

[0269] 53. A cell according to any one of embodiments 43 to 52, wherein the cell is selected from the group consisting of microbial, plant or animal cells, preferably the microbe is a bacterium, fungus or yeast, preferably the plant is a rice, cotton, rapeseed, soybean, maize or corn plant, preferably the animal is an insect, fish, bird or non-human mammal.

[0270] 54. A host cell according to embodiment 53, wherein the cell is an Escherichia coli cell.

[0271] 55. A cell according to any one of embodiments 43 to 54, wherein the cell comprises a catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that is at least partially inactivated, the monosaccharide, disaccharide or oligosaccharide being involved in and / or required for fucosyllactose synthesis.

[0272] 56. A cell according to any one of embodiments 43 to 55, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose and / or difucosyllactose.

[0273] 57. A method for producing fucosyllactose, comprising the steps of:

[0274] a) providing a cell according to any one of embodiments 43 to 56,

[0275] b) culturing the cell in a medium under conditions allowing production of the fucosyllactose.

[0276] c) Isolate the fucosyllactose from the culture.

[0277] 58. Use of a membrane protein selected from the group of membrane proteins as defined in any one of embodiments 1 to 31 in the fermentative production of fucosyllactose.

[0278] 59. Use of a cell according to any one of embodiments 43 to 56 in a method for producing fucosyllactose.

[0279] 60. Use of a cell according to embodiment 59, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose and / or difucosyllactose.

[0280] Furthermore, the present invention relates to the following preferred specific embodiments:

[0281] 1. A method for producing fucosyllactose by genetically modified cells, comprising the following steps:

[0282] - Providing cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a guanosine diphosphate fucose (GDP-fucose) donor to a lactose acceptor to synthesize fucosyllactose,

[0283] - The cells further comprise: i) modified expression of an endogenous membrane protein that enables and / or enhances fucosyllactose transport and / or ii) expression of a heterologous membrane protein that enables and / or enhances fucosyllactose transport, and wherein the membrane protein: i) is selected from the group of membrane proteins comprising any one of the PFAM domains found by searching the genomic neighborhood of the GT10 and GT11 fucosyltransferase families, the GT10 and GT11 fucosyltransferase families having interpro numbers IPR001503 and IPR002516 as defined in InterPro 75.0 released on July 4, 2019, wherein the genomic neighborhood window size is 14 genes before and 14 genes after each fucosyltransferase, and wherein the membrane protein does not belong to the SET family, or ii) is selected from the group consisting of: membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or functional homologs or functional fragments of any one of the membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or sequences having at least 80% sequence identity to any one of the membrane proteins having SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218,

[0284] - Culturing the cells in a culture medium under conditions allowing the production of the desired fucosyllactose,

[0285] - Preferably isolating fucosyllactose from the culture.

[0286] 2. The method according to embodiment 1, wherein the membrane protein is selected from the group consisting of:

[0287] a) Carriers;

[0288] b) Transporters driven by P-P bond hydrolysis;

[0289] c) β-barrel porins;

[0290] d) Cotransporters;

[0291] e) Putative transporters; and

[0292] f) Phosphotransferase-driven group translocation proteins.

[0293] 3. A method for producing fucosyllactose by genetically modified cells, comprising the steps of:

[0294] - Providing cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose,

[0295] - The cells further comprise: i) modified expression of an endogenous membrane protein that allows and / or enhances the transport of fucosyllactose and / or ii) expression of a heterologous membrane protein that allows and / or enhances the transport of fucosyllactose, and wherein the membrane protein is selected from the group consisting of:

[0296] a) Carriers, and wherein the membrane protein does not belong to the SET family;

[0297] b) Transporters driven by P-P bond hydrolysis;

[0298] c) β-barrel porins;

[0299] d) Cotransporters;

[0300] e) Putative transporters; and

[0301] f) Phosphotransferase-driven group translocation proteins,

[0302] - Culturing the cells in a culture medium under conditions allowing the production of the desired fucosyllactose,

[0303] - Preferably isolating fucosyllactose from the culture.

[0304] 4. The method according to any one of embodiments 2 or 3, wherein the transporter is selected from the group consisting of TCDB classes 2.A.1.1, 2.A.1.12, 2.A.1.15, 2.A.1.2, 2.A.1.3, 2.A.1.36, 2.A.1.38, 2.A.1.46, 2.A.1.68, 2.A.1.7, 2.A.1.81, 2.A.123, 2.A.2, 2.A.21, 2.A.58, 2.A.6.3, 2.A.66, 2.A.7.1 as defined by TCDB.org and released on June 17, 2019.

[0305] 5. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the group consisting of TCDB classes 3.A.1.1, 3.A.1.2, 3.A.1.10, 3.A.1.11, and 3.A.1.5 as defined by TCDB.org and released on June 17, 2019.

[0306] 6. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from TCDB classes 1.B.3.1 and 1.B.18 as defined by TCDB.org and released on June 17, 2019.

[0307] 7. The method according to any one of embodiments 2 or 3, wherein the secondary transporter is selected from TCDB class 8.A.3 as defined by TCDB.org and released on June 17, 2019.

[0308] 8. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the group consisting of TCDB classes 9.B.14 and 9.B.158 as defined by TCDB.org and released on June 17, 2019.

[0309] 9. The method according to any one of embodiments 2 or 3, wherein the phosphotransferase-driven group translocase is selected from the group consisting of TCDB classes 4.A.1.1 and 4.A.4.1 as defined by TCDB.org and released on June 17, 2019.

[0310] 10. The method according to any one of embodiments 2 or 3, wherein the transporter is selected from the group consisting of eggnog families 05BZS, 05C0R, 05C2C, 05CT4, 05CXP, 05CZQ, 05D94, 05DXI, 05E5M, 05E5W, 05E8G, 05EAM, 05EDR, 05EGZ, 05F9N, 05JHE, 05PSV, 05W2Y, 05W3H, 05XJ5, 070Q9, 07CWC, 07QF7, 07QNK, 07RBJ, 07RJ1, 07T5E, 07VQ3, 0814C, 088QT, 08H15, 08N8A, 08SC4, 08Z4Q as defined by eggnogdb 1.0.2 released on November 3, 2017.

[0311] 11. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the group consisting of eggnog families 05BZ1, 05CJ1, 05EY8, 05HAC, 05DMK, 05DFW, 05MFV, 07FKK, 07R5U, 07V1T, 08IJ9, 08JQ7, 172T7 as defined by eggnogdb 1.0.2 released on November 3, 2017.

[0312] 12. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from the group consisting of eggnog families 05DAY, 08KDD as defined by eggnogdb 1.0.2 released on November 3, 2017.

[0313] 13. The method according to any one of embodiments 2 or 3, wherein the secondary transporter is selected from the group consisting of eggnog family 07SYR as defined by eggnogdb 1.0.2 released on November 3, 2017.

[0314] 14. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the group consisting of eggnog families 05CRE, 05GWF, 06N3A as defined by eggnogdb 1.0.2 released on November 3, 2017.

[0315] 15. The method according to any one of embodiments 2 or 3, wherein the phosphotransferase-driven group translocase is selected from the group consisting of eggnog families 05CI1 and 05VI0 as defined by eggnogdb 1.0.2 released on November 3, 2017.

[0316] 16. The method according to any one of embodiments 2 or 3, wherein the transporter is selected from the PFAM list PF00083, PF00474, PF00873, PF00893, PF01895, PF01943, PF02690, PF03083, PF04193, PF05977, PF07690, PF07690, PF13347, PF13440, and PF14667 as defined by Pfam 32.0 released in September 2018.

[0317] 17. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the PFAM list PF00005, PF00532, PF00664, PF01061, PF08352, PF14524, PF13407, PF13416, and PF17912 as defined by Pfam 32.0 released in September 2018.

[0318] 18. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from the PFAM list PF02264, PF02563, PF10531, and PF18412 as defined by Pfam 32.0 released in September 2018.

[0319] 19. The method according to any one of embodiments 2 or 3, wherein the secondary transporter is selected from the PFAM list PF13807 and PF02706 as defined by Pfam 32.0 released in September 2018.

[0320] 20. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the PFAM list PF01578, PF03932, PF05140, and PF11045 as defined by Pfam 32.0 released in September 2018.

[0321] 21. The method according to any one of embodiments 2 or 3, wherein the phosphotransferase-driven group translocase is selected from the PFAM list PF00367, PF00358, PF02378, and PF03829 as defined by Pfam 32.0 released in September 2018.

[0322] 22. The method according to any one of embodiments 2 or 3, wherein the transporter is selected from the InterPro list IPR000390, IPR001036, IPR001411, IPR001734, IPR001927, IPR002797, IPR003663, IPR003841, IPR004316, IPR004633, IPR004638, IPR004734, IPR004812, IPR005275, IPR005828, IPR005829, IPR006603, IPR010290, IPR011701, IPR020846, IPR023008, IPR023721, IPR023722, IPR026022, IPR027417, IPR027463, IPR029303, IPR032896, IPR036259, IPR038078, IPR038377, IPR039672 as defined by InterPro 75.0 released on July 4, 2019.

[0323] 23. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from the InterPro list IPR000412, IPR001734, IPR001761, IPR003439, IPR003593, IPR005829, IPR005978, IPR005981, IPR006059, IPR006060, IPR006061, IPR008995, IPR011527, IPR011701, IPR013456, IPR013525, IPR013563, IPR015851, IPR015855, IPR017871, IPR019554, IPR020846, IPR025997, IPR026266, IPR027417, IPR028082, IPR029439, IPR033893, IPR036259, IPR036640, IPR038377, IPR039421 and IPR040582 as defined by InterPro 75.0 released on July 4, 2019.

[0324] 24. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from the InterPro list IPR003192, IPR003715, IPR019554, IPR023738, IPR036998 and IPR040716 as defined by InterPro 75.0 released on July 4, 2019.

[0325] 25. A method according to any one of embodiments 2 or 3, wherein the auxiliary transporter is selected from the interpro lists IPR003856, IPR020846, IPR027417, IPR032807, and IPR036259 as defined in InterPro 75.0 released on July 4, 2019.

[0326] 26. A method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from the interpro lists IPR002541, IPR003439, IPR003593, IPR004316, IPR005627, IPR006603, IPR007816, IPR017871, IPR020368, IPR020846, IPR023648, IPR027417, IPR036259, and IPR036822 as defined in InterPro 75.0 released on July 4, 2019.

[0327] 27. A method according to any one of embodiments 2 or 3, wherein the phosphotransferase-driven group translocation protein is selected from the interpro lists IPR001127, IPR001996, IPR003352, IPR004716, IPR010974, IPR011055, IPR013013, IPR018113, IPR018454, IPR036665, and IPR036878 as defined in InterPro 75.0 released on July 4, 2019.

[0328] 28. A method according to any one of embodiments 2 or 3, wherein the transporter membrane protein is selected from: MdfA from Escherichia coli K12 MG1655 having SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 having SEQ ID NO 06, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 42, a wzx-like protein from Chitinophaga sp. CF118 having SEQ ID NO 58, Prevotella ruminicola (AR32) having SEQ ID NO 66, Lactococcus raffinolactis (ATCC 43920) having SEQ ID NO 64, or an Acinetobacter soli DSM 25329 wzx-like protein having SEQ ID NO 62, or a functional homolog or functional fragment of any of the above transporter membrane proteins, or a sequence having at least 80% sequence identity to any one of the MdfA, IceT, Blon_2331, Blon_2332 membrane proteins or wzx-like membrane proteins having SEQ ID NOs 02, 06, 40, 42, 58, 66, 64 or 62, respectively.

[0329] 29. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from: lmrA from Lactococcus lactis strain SRCM103457 having SEQ ID NO 28, LpsE membrane protein from Mycobacterium sphaeroides DSM 2875 having SEQ ID NO 70 or 74, LpsE membrane protein from Flavobacterium spartans having SEQ ID NO 68 or 72, TolC from Candidatus Planktophila sulfonica having SEQ ID NO 76, MsbA from Pedobacter ginsengisoli having SEQ ID NO 82 or MsbA from Verrucomicrobia bacterium CG1_02_43_26 having SEQ ID NO 84, malE from Escherichia coli K-12 MG1655 having SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 having SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 having SEQ ID NO 214, xylF from Escherichia coli K-12 MG1655 having SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 having SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins, or a sequence having at least 80% sequence identity with any of the lmrA, LpsE, TolC, MsbA, malE, malK, araF, xylF or ytfQ membrane proteins having SEQ ID NO 28, 70, 74, 68, 72, 76, 82, 84, 206, 208, 214, 216 or 218, respectively.

[0330] 30. The method according to any one of embodiments 2 or 3, wherein the β-barrel porin is selected from: Wza from Escherichia coli K12 MG1655 having SEQ ID NO 34 or lamB from Escherichia coli K12 MG1655 having SEQ ID NO 204, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity with the Wza or lamB membrane protein having SEQ ID NO 34 or 204, respectively.

[0331] 31. The method according to any one of embodiments 2 or 3, wherein the secondary transporter is selected from: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) having SEQ ID NO 88, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity with the Wzc membrane protein having SEQ ID NO 88.

[0332] 32. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from: CutC from Clostridium species CAG:1013 having SEQ ID NO 90, from Odoribacter splanchnicus DSM 20712 having SEQ ID NO 92, from Inquilinus limosus species PDC51 having SEQ ID NO 94, or from Prevotella intermedia ATCC 25611 (DSM 20706) having SEQ ID NO 96, or a functional homolog or functional fragment of any of the CutC membrane proteins, or a sequence having at least 80% sequence identity with any of the CutC membrane proteins having SEQ ID NO 90, 92, 94, or 96, respectively.

[0333] 33. The method according to any one of embodiments 2 or 3, wherein the phosphotransferase-driven group translocation protein is selected from: nagE from Escherichia coli K12 MG1655 having SEQ ID NO 210, srlB from Escherichia coli K12 MG1655 having SEQ ID NO 212, or a functional homolog or functional fragment of any of the nagE or srlB membrane proteins, or a sequence having at least 80% sequence identity with any of the nagE or srlB membrane proteins having SEQ ID NO 210 or 212, respectively.

[0334] 34. The method according to any one of embodiments 2 or 3, wherein the transporter membrane protein is selected from: MdfA from Escherichia coli K12 MG1655 having SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 having SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 having SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 having SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 having SEQ ID NO 10, YdhC from Escherichia coli K12 MG1655 having SEQ ID NO 12, YbdA from Escherichia coli K12 MG1655 having SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 having SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 having SEQ ID NO 18, YebQ from Escherichia coli K12 MG1655 having SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 having SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 having SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 having SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 having SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 having SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 50, CDT2 from Neurospora crassa OR74A having SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 having SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 having SEQ ID NO 58, Wzx from Eubacterium sp. CAG:581 having SEQ ID NO 60Wzx from Agrobacterium tumefaciens (DSM 25329) having SEQ ID NO 62, Wzx from Lactococcus raffinolactis (ATCC 43920) having SEQ ID NO 64, Wzx from Prevotella ruminicola (AR32) having SEQ ID NO 66, NAPO from Brachyspira hampsonii P280 / 1 having SEQ ID NO 86, NAm from Actinobacillus suis (DSM 20639) having SEQ ID NO 98, NAm from Ruminococcus gnavus having SEQ ID NO 100, NAm from Brevibacterium species 314Chir4.1 having SEQ ID NO 102, Nap from Niabelladrilacis (DSM25811) having SEQ ID NO 104, Nap from Saccharicrinis fermentans (DSM9555) having SEQ ID NO 106, mdtD from Citrobacter freundii MGH152 having SEQ ID NO 108, mdtD from Citrobacter werkmanii NBRC 105721 having SEQ ID NO 110, mdtD from Citrobacter amalonaticus having SEQ ID NO 112, mdtD from Klebsiella oxytoca having SEQ ID NO 114, mdtD from Escherichia albertii B156 having SEQ ID NO 116, yegB from Salmonella enterica subsp. salamae having SEQ ID NO 118, mdtD from Klebsiella pneumoniae 30684 / NJST258_2 having SEQ ID NO 120, Tcr_1_D38215 from Klebsiella pneumoniae having SEQ ID NO 122, mdtD from Citrobacter farmeri having SEQ ID NO 124, Cmr from Prevotella regensburgensis (ATCC43003) having SEQ ID NO 126, MdfA from Cronobacter muytjensii having SEQ ID NO 128, MdfA from Klebsiella oxytoca having SEQ ID NO 130, MFS from Citrobacter koseri having SEQ ID NO 132, MdfA from Escherichia marmotae having SEQ ID NO 134, Cmr from Shigella flexneri having SEQ ID NO 136, MdfA from Salmonella enterica subsp. salamae having SEQ ID NO 138, Cmr from Citrobacter youngae (ATCC 29220) having SEQ ID NO 140, MdfA from Citrobacter freundii having SEQ ID NO 142MdfA from Enterobacter kobei with SEQ ID NO 144, MdfA from an Enterobacter species with SEQ ID NO 146, MdfA from Lelliottia species WB101 with SEQ ID NO 148, MdfA from Enterobacter ludwigii EcWSU1 with SEQ ID NO 150, thaumatin-like protein from Actinoplanes utahensis with SEQ ID NO 152, thaumatin-like protein from Chitinophagaceae bacterium PMG_246 with SEQ ID NO 154, thaumatin-like protein from Rhizobium species PDC82 with SEQ ID NO 156, thaumatin-like protein from Kineococcus rhizophilus (DSM 19711) with SEQ ID NO 158, thaumatin-like protein from Morganella morganii IS15 with SEQ ID NO 160, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078) with SEQ ID NO 162, thaumatin-like protein from Bradyrhizobium sp. BTAi1 with SEQ ID NO 164, thaumatin-like protein from Bradyrhizobium japonicum USDA110 with SEQ ID NO 166, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10 with SEQ ID NO 168, thaumatin-like protein from Aquaspirillum itersonii with SEQ ID NO 170, thaumatin-like protein from Flavobacterium bacterium MS024-2A with SEQ ID NO 172, rnd-like protein from Sinorhizobium meliloti WSM419 with SEQ ID NO 182, arabinose efflux protein from Azospirillum brasilense LMG04375 with SEQ ID NO 184, or a functional homolog or functional fragment of any of the above transporter membrane proteinsor a protein having an amino acid sequence with at least 80% sequence identity to any of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, sweet-like or arabinose efflux membrane proteins respectively having SEQ ID NO 02, 06, 04, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 32, 38, 40, 42, 46, 48, 50, 52, 54, 58, 60, 62, 64, 66, 86, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 182 or 184.,

[0335] 35. The method according to any one of embodiments 2 or 3, wherein the P-P bond hydrolysis-driven transporter is selected from: LmrA from Lactococcus lactis strain SRCM103457 having SEQ ID NO 28, OppF from Escherichia coli strain K12 MG1655 having SEQ ID NO 30, Wzk from Helicobacter pylori (strain ATCC700392 / 26695) having SEQ ID NO 36, Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 44, LpsE from Flavobacterium spartans having SEQ ID NO 68 or 72, LpsE from Mycococcus globosus DSM 2875 having SEQ ID NO 70 or 74, TolC from Candidatus Planktophila sulfonica having SEQ ID NO 76, TolC from Butyrivibrio hungatei XBD2006 having SEQ ID NO 78, MsbA from Roseburia intestinalis CAG:13 having SEQ ID NO 80, MsbA from Pedobacter ginsengisoli having SEQ ID NO 82, MsbA from Verrucomicrobia bacterium CG1_02_43_26 having SEQ ID NO 84, Wzm from Rhizobium sp. Root149 having SEQ ID NO 174, Wzm from Azospirillum brasilense LMG 04375 having SEQ ID NO 176, Wzm from Escherichia coli 113303 having SEQ ID NO 196, Wzt from Rhizobium sp. Root149 having SEQ ID NO 178, Wzt from Azospirillum brasilense LMG 04375 having SEQ ID NO 180, Wzt from Escherichia coli 113303 having SEQ ID NO 194, Nodj from Bradyrhizobium japonicum USDA110 having SEQ ID NO 188 or 190, malE from Escherichia coli K-12 MG1655 having SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 having SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 having SEQ ID NO 214, xylF from Escherichia coli K-12 MG1655 having SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 having SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins,or a protein having an amino acid sequence with at least 80% sequence identity to any one of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt, Nodj, malE, malK, araF, xylF, or ytfQ membrane proteins having SEQ ID NO 28, 30, 36, 44, 68, 72, 70, 74, 76, 78, 80, 82, 84, 174, 176, 196, 178, 180, 194, 188, 190, 206, 208, 214, 216, or 218, respectively.,

[0336] 36. The method according to any one of embodiments 2 or 3, wherein the putative transporter is selected from: a cytochrome C biogenesis protein from Helicobacter pylori having SEQ ID NO 56, CutC from Clostridium species CAG:1013 having SEQ ID NO 90, CutC from Odoribacter splanchnicus DSM 20712 having SEQ ID NO 92, CutC from Flavobacterium species PDC51 having SEQ ID NO 94, CutC from Prevotella intermedia ATCC 25611 (DSM 20706) having SEQ ID NO 96, ybjM from Escherichia coli K12 MG1655 having SEQ ID NO 190, ybjM from Enterobacteriaceae bacterium ENNIH1 having SEQ ID NO 192, or a functional homolog or functional fragment of any of the above putative transporters, or a protein having an amino acid sequence with at least 80% sequence identity to any one of the CytC, CutC, or ybjM membrane proteins having SEQ ID NO 56, 90, 92, 94, 96, 190, or 192, respectively.

[0337] 37. A method for producing fucosyllactose in a genetically modified cell, comprising the steps of:

[0338] - providing a cell capable of producing fucosyllactose, the cell comprising at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose,

[0339] - the cell further comprising: i) modified expression of an endogenous membrane protein that enables and / or enhances fucosyllactose transport and / or ii) expression of a heterologous membrane protein that enables and / or enhances fucosyllactose transport, and wherein the membrane protein is selected from the group of membrane proteins consisting of:

[0340] Transporter membrane proteins: MdfA from Escherichia coli K12 MG1655 with SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 with SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 with SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 with SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 with SEQ ID NO 10, YdhC from Escherichia coli K12 MG1655 with SEQ ID NO 12, YbdA from Escherichia coli K12 MG1655 with SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 with SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 with SEQ ID NO 18, YebQ from Escherichia coli K12 MG1655 with SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 with SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 with SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 with SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 with SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 with SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with SEQ ID NO 50, CDT2 from Neurospora crassa OR74A with SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 with SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 with SEQ ID NO 58, Wzx from Eubacterium sp. CAG:581 with SEQ ID NO 60, Wzx from Agrobacterium tumefaciens (DSM25329) with SEQ ID NO 62Wzx from Lactococcus raffinolactis (ATCC 43920) with SEQ ID NO 64, Wzx from Prevotella ruminicola (AR32) with SEQ ID NO 66, NAPO from Brachyspira hampsonii P280 / 1 with SEQ ID NO 86, NAm from Actinobacillus suis (DSM 20639) with SEQ ID NO 98, NAm from Ruminococcus gnavus with SEQ ID NO 100, NAm from Brevibacterium sp. 314Chir4.1 with SEQ ID NO 102, Nap from Niabella drilacis (DSM 25811) with SEQ ID NO 104, Nap from Saccharicrinis fermentans (DSM 9555) with SEQ ID NO 106, mdtD from Citrobacter freundii MGH152 with SEQ ID NO 108, mdtD from Citrobacter werkmanii NBRC 105721 with SEQ ID NO 110, mdtD from Citrobacter amalonaticus with SEQ ID NO 112, mdtD from Klebsiella oxytoca with SEQ ID NO 114, mdtD from Escherichia albertii B156 with SEQ ID NO 116, yegB from Salmonella enterica subsp. salamae with SEQ ID NO 118, mdtD from Klebsiella pneumoniae 30684 / NJST258_2 with SEQ ID NO 120, Tcr_1_D38215 from Klebsiella pneumoniae with SEQ ID NO 122, mdtD from Citrobacter farmeri with SEQ ID NO 124, Cmr from Prevotella regensburgensis (ATCC 43003) with SEQ ID NO 126, MdfA from Cronobacter muytjensii with SEQ ID NO 128, MdfA from Klebsiella oxytoca with SEQ ID NO 130, MFS from Citrobacter koseri with SEQ ID NO 132, MdfA from Escherichia marmotae with SEQ ID NO 134, Cmr from Shigella flexneri with SEQ ID NO 136, MdfA from Salmonella enterica subsp. salamae with SEQ ID NO 138, Cmr from Citrobacter youngae (ATCC 29220) with SEQ ID NO 140, MdfA from Citrobacter freundii with SEQ ID NO 142, MdfA from Enterobacter kobei with SEQ ID NO 144MdfA from an Enterobacter species having SEQ ID NO 146, MdfA from a Lelliottia species WB101 having SEQ ID NO 148, MdfA from Enterobacter ludwigii EcWSU1 having SEQ ID NO 150, thaumatin-like protein from Actinoplanes utahensis having SEQ ID NO 152, thaumatin-like protein from a Chitinophagaceae bacterium PMG_246 having SEQ ID NO 154, thaumatin-like protein from a Rhizobium species PDC82 having SEQ ID NO 156, thaumatin-like protein from Kineococcus rhizophilus (DSM 19711) having SEQ ID NO 158, thaumatin-like protein from Morganella morganii IS15 having SEQ ID NO 160, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078) having SEQ ID NO 162, thaumatin-like protein from Bradyrhizobium sp. BTAi1 having SEQ ID NO 164, thaumatin-like protein from Bradyrhizobium japonicum USDA110 having SEQ ID NO 166, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10 having SEQ ID NO 168, thaumatin-like protein from Aquaspirillum itersonii having SEQ ID NO 170, thaumatin-like protein from a Flavobacterium bacterium MS024-2A having SEQ ID NO 172, an rnd-like protein from Sinorhizobium meliloti WSM419 having SEQ ID NO 182, an arabinose efflux protein from Azospirillum brasilense LMG 04375 having SEQ ID NO 184, or a functional homolog or functional fragment of any of the above transporter membrane proteins,or a protein having an amino acid sequence with at least 80% sequence identity to any of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, class-sweet or arabinose efflux membrane proteins respectively having SEQ ID NO 02, 06, 04, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 32, 38, 40, 42, 46, 48, 50, 52, 54, 58, 60, 62, 64, 66, 86, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 182 or 184; and,

[0341] P-P bond hydrolysis-driven transporters: LmrA from Lactococcus lactis strain SRCM103457 with SEQ ID NO 28, OppF from Escherichia coli strain K12 MG1655 with SEQ ID NO 30, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695) with SEQ ID NO 36, Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with SEQ ID NO 44, LpsE from Flavobacterium spartans with SEQ ID NO 68 or 72, LpsE from Myxococcus fulvus DSM 2875 with SEQ ID NO 70 or 74, TolC from Candidatus Planktophila sulfonica with SEQ ID NO 76, TolC from Butyrivibrio hungatei XBD2006 with SEQ ID NO 78, MsbA from Roseburia intestinalis CAG:13 with SEQ ID NO 80, MsbA from Pedobacter ginsengisoli with SEQ ID NO 82, MsbA from Verrucomicrobia bacterium CG1_02_43_26 with SEQ ID NO 84, Wzm from Rhizobium sp. Root149 with SEQ ID NO 174, Wzm from Azospirillum brasilense LMG 04375 with SEQ ID NO 176, Wzm from Escherichia coli 113303 with SEQ ID NO 196, Wzt from Rhizobium sp. Root149 with SEQ ID NO 178, Wzt from Azospirillum brasilense LMG 04375 with SEQ ID NO 180, Wzt from Escherichia coli 113303 with SEQ ID NO 194, Nodj from Bradyrhizobium japonicum USDA110 with SEQ ID NO 188 or 190, malE from Escherichia coli K-12 MG1655 with SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 with SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 with SEQ ID NO 214, xylF from Escherichia coli K-12 MG1655 with SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 with SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporters,or a protein having an amino acid sequence with at least 80% sequence identity to any one of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt, Nodj, malE, malK, araF, xylF, or ytfQ membrane proteins having SEQ ID NOs 28, 30, 36, 44, 68, 72, 70, 74, 76, 78, 80, 82, 84, 174, 176, 196, 178, 180, 194, 188, 190, 206, 208, 214, 216, or 218, respectively; and,

[0342] β-barrel pore-forming membrane protein: Wza from Escherichia coli K12 MG1655 having SEQ ID NO 34 or lamB from Escherichia coli K12 MG1655 having SEQ ID NO 204, or a functional homolog or functional fragment of any one of the Wza or lamB proteins, or a sequence having at least 80% sequence identity to any one of the Wza or lamB membrane proteins having SEQ ID NO 34 or 204, respectively; and

[0343] Secondary transporter: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM3109 / JCM 10099) having SEQ ID NO 88, or a functional homolog or functional fragment thereof, or a sequence having at least 80% sequence identity to the Wzc membrane protein having SEQ ID NO 88;

[0344] Putative transporter: Cytochrome C biogenesis protein from Helicobacter pylori having SEQ ID NO 56, CutC from Clostridium species CAG:1013 having SEQ ID NO 90, CutC from Odoribacter splanchnicus DSM 20712 having SEQ ID NO 92, CutC from Flavobacterium songnenense species PDC51 having SEQ ID NO 94, CutC from Prevotella intermedia ATCC 25611 (DSM 20706) having SEQ ID NO 96, ybjM from Escherichia coli K12 MG1655 having SEQ ID NO 190, ybjM from Enterobacteriaceae bacterium ENNIH1 having SEQ ID NO 192, or a functional homolog or functional fragment of any one of the above putative transporters, or a protein having an amino acid sequence with at least 80% sequence identity to any one of the CytC, CutC, or ybjM membrane proteins having SEQ ID NOs 56, 90, 92, 94, 96, 190, or 192, respectively; and

[0345] Phosphate transporter-driven group translocation proteins: nagE from Escherichia coli K12 MG1655 having SEQ ID NO 210, srlB from Escherichia coli K12 MG1655 having SEQ ID NO 212, or a functional homolog or functional fragment of any one of said nagE or srlB membrane proteins, or a sequence having at least 80% sequence identity to any one of said nagE or srlB membrane proteins having SEQ ID NO 210 or 212, respectively.

[0346] 38. A method for producing fucosyllactose according to any one of the foregoing specific embodiments, wherein said membrane protein is a transporter protein involved in the outer membrane transport of a compound across the cell wall.

[0347] 39. A method for producing fucosyllactose according to any one of the foregoing specific embodiments, the method further comprising at least one of the following steps:

[0348] i) adding a lactose feed to the culture medium, comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250 mL to 10,000 m 3 , preferably added in a continuous manner, and preferably such that the final volume of the culture medium is no more than three times, preferably no more than twice, more preferably less than 2 times the volume of the culture medium before adding said lactose feed;

[0349] ii) adding a lactose feed to the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days through a feed solution;

[0350] iii) adding a lactose feed to the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days through a feed solution, and wherein the concentration of said lactose feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein preferably the pH value of said solution is set to 3 to 7 and wherein preferably the temperature of said feed solution is maintained at 20 °C to 80 °C;

[0351] The method produces a fucosyllactose concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the culture medium.

[0352] 40. The method according to embodiment 39, wherein lactose feeding is achieved by adding lactose at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration > 300 mM from the start of the culture.

[0353] 41. The method according to any one of embodiments 39 or 40, wherein the lactose feeding is achieved by adding a certain concentration of lactose to the culture medium such that a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained during the production phase of the entire culture.

[0354] 42. The method according to any one of embodiments 39, 40 or 41, wherein the host cells are cultured for at least about 60, 80, 100 or about 120 hours or in a continuous manner.

[0355] 43. The method according to any one of embodiments 39 to 42, wherein a carbon and energy source is also added, preferably sucrose, glucose, fructose, glycerol, maltose, maltodextrin, trehalose, polyol, starch, succinate, malate, pyruvate, lactate, ethanol, citrate, lactose, preferably continuously added to the culture medium, preferably together with lactose.

[0356] 44. The method according to any one of embodiments 39 to 43, wherein a first stage of exponential cell growth is provided by adding a carbon substrate, preferably glucose or sucrose, to the culture medium before adding lactose to the culture medium in the second stage.

[0357] 45. The method according to any one of embodiments 1 to 44, wherein the method produces a mixture of fucosyllactose.

[0358] 46. The method according to any one of embodiments 1 to 45, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose and / or difucosyllactose.

[0359] 47. A method according to any one of embodiments 1 to 46, wherein the genetically modified cell is selected from the group consisting of microbial, plant or animal cells, preferably the microbe is a bacterium, fungus or yeast, preferably the plant is a rice, cotton, rapeseed, soybean, maize or corn plant, preferably the animal is an insect, fish, bird or non-human mammal.

[0360] 48. A method according to embodiment 47, wherein the cell is an Escherichia coli cell.

[0361] 49. A host cell genetically modified for the production of fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose,

[0362] - the cell further comprises: i) a modified expression of an endogenous membrane protein that enables and / or enhances the transport of fucosyllactose and / or ii) an expression of a heterologous membrane protein that enables and / or enhances the transport of fucosyllactose, and wherein the membrane protein: i) is selected from the group of membrane proteins that contain any one of the PFAM domains found by searching the genomic neighborhood of the GT10 and GT11 fucosyltransferase families, the GT10 and GT11 fucosyltransferase families having interpro numbers IPR001503 and IPR002516 as defined in InterPro 75.0 released on July 4, 2019, wherein the genomic neighborhood window size is 14 genes before and 14 genes after each fucosyltransferase, and wherein the membrane protein does not belong to the SET family, or ii) is selected from the group consisting of: membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or functional homologs or functional fragments of any one of the membrane proteins comprising SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218, or sequences having at least 80% sequence identity to any one of the membrane proteins having SEQ ID NOs 204, 206, 208, 210, 212, 214, 216, 218.

[0363] 50. A host cell according to embodiment 49, wherein the membrane protein is selected from the group consisting of:

[0364] a) a porter;

[0365] b) a P-P bond hydrolysis-driven transporter;

[0366] c) a β-barrel porin;

[0367] d) Secondary transporter;

[0368] e) Putative transporter; and

[0369] f) Phosphotransferase-driven group translocation protein.

[0370] 51. A host cell genetically modified for the production of fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose,

[0371] - the cell further comprises: i) a modified expression of an endogenous membrane protein enabling and / or enhancing the transport of fucosyllactose and / or ii) an expression of a heterologous membrane protein enabling and / or enhancing the transport of fucosyllactose, and wherein the membrane protein is selected from the group consisting of:

[0372] a) Carrier, and wherein the membrane protein does not belong to the SET family;

[0373] b) P-P bond hydrolysis-driven transporter;

[0374] c) β-barrel porin;

[0375] d) Secondary transporter;

[0376] e) Putative transporter; and

[0377] f) Phosphotransferase-driven group translocation protein.

[0378] 52. The host cell according to any one of embodiments 50 or 51, wherein the membrane protein is selected from the group of membrane proteins as defined in any one of embodiments 4 to 38.

[0379] 53. A host cell genetically modified for the production of fucosyllactose according to any one of embodiments 50 or 51, wherein the porter membrane protein is selected from: MdfA from Escherichia coli K12 MG1655 having SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 having SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 having SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 having SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 having SEQ ID NO 10, YdhC from Escherichia coli K12 MG1655 having SEQ ID NO 12, YbdA from Escherichia coli K12 MG1655 having SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 having SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 having SEQ ID NO 18, YebQ from Escherichia coli K12 having SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 having SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 having SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 having SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 having SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 having SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 50, CDT2 from Neurospora crassa OR74A having SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 having SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 having SEQ ID NO 58, Wzx from Eubacterium sp. CAG:581 having SEQ ID NO 60,Wzx from Agrobacterium tumefaciens (DSM 25329) with SEQ ID NO 62, Wzx from Lactococcus raffinolactis (ATCC 43920) with SEQ ID NO 64, Wzx from Prevotella ruminicola (AR32) with SEQ ID NO 66, NAPO from Brachyspira hampsonii P280 / 1 with SEQ ID NO 86, NAm from Actinobacillus suis (DSM 20639) with SEQ ID NO 98, NAm from Ruminococcus gnavus with SEQ ID NO 100, NAm from Brevibacterium species 314Chir4.1 with SEQ ID NO 102, Nap from Niabella drilacis (DSM 25811) with SEQ ID NO 104, Nap from Saccharicrinis fermentans (DSM 9555) with SEQ ID NO 106, mdtD from Citrobacter freundii MGH152 with SEQ ID NO 108, mdtD from Citrobacter werkmanii NBRC 105721 with SEQ ID NO 110, mdtD from Citrobacter amalonaticus with SEQ ID NO 112, mdtD from Klebsiella oxytoca with SEQ ID NO 114, mdtD from Escherichia albertii B156 with SEQ ID NO 116, yegB from Salmonella enterica subsp. salamae with SEQ ID NO 118, mdtD from Klebsiella pneumoniae 30684 / NJST258_2 with SEQ ID NO 120, Tcr_1_D38215 from Klebsiella pneumoniae with SEQ ID NO 122, mdtD from Citrobacter farmeri with SEQ ID NO 124, Cmr from Prevotella regensburgensis (ATCC 43003) with SEQ ID NO 126, MdfA from Cronobacter muytjensii with SEQ ID NO 128, MdfA from Klebsiella oxytoca with SEQ ID NO 130, MFS from Citrobacter koseri with SEQ ID NO 132, MdfA from Escherichia marmotae with SEQ ID NO 134, Cmr from Shigella flexneri with SEQ ID NO 136, MdfA from Salmonella enterica subsp. salamae with SEQ ID NO 138, Cmr from Citrobacter youngae (ATCC 29220) with SEQ ID NO 140, MdfA from Citrobacter freundii with SEQ ID NO 142MdfA from Enterobacter kobei with SEQ ID NO 144, MdfA from an Enterobacter species with SEQ ID NO 146, MdfA from Lelliottia species WB101 with SEQ ID NO 148, MdfA from Enterobacter ludwigii EcWSU1 with SEQ ID NO 150, thaumatin-like protein from Actinoplanes utahensis with SEQ ID NO 152, thaumatin-like protein from Chitinophagaceae bacterium PMG_246 with SEQ ID NO 154, thaumatin-like protein from Rhizobium species PDC82 with SEQ ID NO 156, thaumatin-like protein from Kineococcus rhizophilus (DSM 19711) with SEQ ID NO 158, thaumatin-like protein from Morganella morganii IS15 with SEQ ID NO 160, thaumatin-like protein from Dermatophilus obscurus (strain ATCC 25078) with SEQ ID NO 162, thaumatin-like protein from Bradyrhizobium species BTAi1 with SEQ ID NO 164, thaumatin-like protein from Bradyrhizobium japonicum USDA110 with SEQ ID NO 166, thaumatin-like protein from Xanthomonas campestris pv. vesicatoria strain 85-10 with SEQ ID NO 168, thaumatin-like protein from Aquaspirillum itersonii with SEQ ID NO 170, thaumatin-like protein from Flavobacterium bacterium MS024-2A with SEQ ID NO 172, rnd-like protein from Sinorhizobium meliloti WSM419 with SEQ ID NO 182, arabinose efflux protein from Azospirillum brasilense LMG 04375 with SEQ ID NO 184, or a functional homolog or functional fragment of any of the above transporter membrane proteins,or a protein having an amino acid sequence with at least 80% sequence identity to any of the MdfA, IceT, YnfM, Yhhs, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr, FucP, WzxE, EmrE, Wzx, Blon_2331, Blon_2232, Blon_0247, Blon_0245, Blon_0345, NAPO, NAm, Nap, mdtD, YegB, Tcr_1_D38215, cmr, MFS, CDT2, rnd, class sweet or arabinose efflux membrane proteins respectively having SEQ ID NO 02, 06, 04, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 32, 38, 40, 42, 46, 48, 50, 52, 54, 58, 60, 62, 64, 66, 86, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 182 or 184.,

[0380] 54. A host cell according to any one of embodiments 50 or 51, wherein the P-P bond hydrolysis-driven transporter is selected from: LmrA from Lactococcus lactis strain SRCM103457 having SEQ ID NO 28, OppF from Escherichia coli strain K12 MG1655 having SEQ ID NO 30, Wzk from Helicobacter pylori (strain ATCC 700392 / 26695) having SEQ ID NO 36, Blon_2475 from Bifidobacterium longum subsp. infantis (strain ATCC 15697) having SEQ ID NO 44, LpsE from Flavobacterium spartans having SEQ ID NO 68 or 72, LpsE from Myxococcus fulvus DSM 2875 having SEQ ID NO 70 or 74, TolC from Candidatus Planktophila sulfonica having SEQ ID NO 76, TolC from Butyrivibrio hungatei XBD2006 having SEQ ID NO 78, MsbA from Roseburia intestinalis CAG:13 having SEQ ID NO 80, MsbA from Pedobacter ginsengisoli having SEQ ID NO 82, MsbA from Verrucomicrobia bacterium CG1_02_43_26 having SEQ ID NO 84, Wzm from Rhizobium sp. Root149 having SEQ ID NO 174, Wzm from Azospirillum brasilense LMG 04375 having SEQ ID NO 176, Wzm from Escherichia coli 113303 having SEQ ID NO 196, Wzt from Rhizobium sp. Root149 having SEQ ID NO 178, Wzt from Azospirillum brasilense LMG 04375 having SEQ ID NO 180, Wzt from Escherichia coli 113303 having SEQ ID NO 194, Nodj from Bradyrhizobium japonicum USDA 110 having SEQ ID NO 188 or 190, malE from Escherichia coli K-12 MG1655 having SEQ ID NO 206, malK from Escherichia coli K-12 MG1655 having SEQ ID NO 208, araF from Escherichia coli K-12 MG1655 having SEQ ID NO 214, xylF from Escherichia coli K-12 MG1655 having SEQ ID NO 216, or ytfQ from Escherichia coli K-12 MG1655 having SEQ ID NO 218, or a functional homolog or functional fragment of any of the above P-P bond hydrolysis-driven transporter membrane proteins,or a protein having an amino acid sequence with at least 80% sequence identity to any one of the LmrA, OppF, Wzk, Blon_2475, LpsE, TolC, MsbA, Wzm, Wzt, Nodj, malE, malK, araF, xylF or ytfQ membrane proteins having SEQ ID NO 28, 30, 36, 44, 68, 72, 70, 74, 76, 78, 80, 82, 84, 174, 176, 196, 178, 180, 194, 188, 190, 206, 208, 214, 216 or 218, respectively.

[0381] 55. The host cell according to any one of specific embodiments 50 or 51, wherein the putative transporter is selected from: cytochrome C biogenesis protein from Helicobacter pylori having SEQ ID NO 56, CutC from Clostridium species CAG:1013 having SEQ ID NO 90, CutC from Odoribacter splanchnicus DSM 20712 having SEQ ID NO 92, CutC from Inquilinus limosus species PDC51 having SEQ ID NO 94, CutC from Prevotella intermedia ATCC 25611 (DSM 20706) having SEQ ID NO 96, ybjM from Escherichia coli K12 MG1655 having SEQ ID NO 190, ybjM from Enterobacteriaceae bacterium ENNIH1 having SEQ ID NO 192, or a functional homolog or functional fragment of any of the above putative transporters, or a protein having an amino acid sequence with 80% sequence identity to any one of the CytC, CutC or ybjM membrane proteins having SEQ ID NO 56, 90, 92, 94, 96, 190 or 192, respectively.

[0382] 56. The host cell according to any one of specific embodiments 50 or 51, wherein the β-barrel porin is selected from: Wza from Escherichia coli K12 MG1655 having SEQ ID NO 34 or lamB from Escherichia coli K12 MG1655 having SEQ ID NO 204, or a functional homolog or functional fragment of any of the Wza or lamB proteins, or a sequence having at least 80% sequence identity to any one of the Wza or lamB membrane proteins having SEQ ID NO 34 or 204, respectively.

[0383] 57. A host cell according to any one of embodiments 50 or 51, wherein the secondary transporter is selected from: Wzc from Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) having SEQ ID NO 88, or a functional homolog or a functional fragment thereof, or a sequence having at least 80% sequence identity with the Wzc membrane protein having SEQ ID NO 88.

[0384] 58. A host cell according to any one of embodiments 50 or 51, wherein the phosphotransferase-driven group translocation protein is selected from: nagE from Escherichia coli K12 MG1655 having SEQ ID NO 210 or srlB from Escherichia coli K12 MG1655 having SEQ ID NO 212, or a functional homolog or a functional fragment of any one of the nagE or srlB membrane proteins, or a sequence having at least 80% sequence identity with any one of the nagE or srlB membrane proteins having SEQ ID NO 210 or 212, respectively.

[0385] 59. A cell according to any one of the foregoing embodiments 49 to 58, wherein the membrane protein is a transporter involved in the outer membrane transport of a compound across the cell wall.

[0386] 60. A cell according to any one of embodiments 49 to 59, wherein the cell is stably cultured in a medium.

[0387] 61. A cell according to any one of embodiments 49 to 60, wherein the cell is selected from the group consisting of microbial, plant or animal cells, preferably the microbe is a bacterium, a fungus or a yeast, preferably the plant is a rice, cotton, rapeseed, soybean, maize or corn plant, preferably the animal is an insect, a fish, a bird or a non-human mammal.

[0388] 62. A host cell according to embodiment 61, wherein the cell is an Escherichia coli cell.

[0389] 63. A cell according to any one of embodiments 49 to 62, wherein the cell comprises a catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that is at least partially inactivated, and the monosaccharide, disaccharide or oligosaccharide is involved in the synthesis of fucosyllactose and / or is required for the synthesis of fucosyllactose.

[0390] 64. A cell according to any one of embodiments 49 to 63, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose.

[0391] 65. A method for producing fucosyllactose, comprising the steps of:

[0392] a) providing a cell according to any one of embodiments 49 to 64,

[0393] b) culturing the cell in a culture medium under conditions allowing the production of said fucosyllactose,

[0394] c) isolating said fucosyllactose from the culture.

[0395] 66. Use of a membrane protein selected from the group of membrane proteins as defined in any one of embodiments 1 to 38 for fucosyllactose transport in the fermentative production of fucosyllactose.

[0396] 67. Use of a cell according to any one of embodiments 49 to 64 for the production of fucosyllactose.

[0397] 68. Use of a cell according to embodiment 67, wherein said fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose.

[0398] The following figures and examples will be used for further illustration and clarification of the present invention and are not intended to limit the present invention. Description of the Drawings

[0399] Figure 1 : CPI expressed as a relative percentage (%) obtained in a growth experiment, using strains expressing a membrane protein having SEQ ID NOs 58 to 96 (excluding SEQ ID NO 90) in TU2, SEQ ID NO 90 in TU3 or SEQ ID NOs 02 to 44 in TU10 and expressing α1,3-fucosyltransferase. Strains with membrane protein SEQ ID NOs 04 to 34 produce 3-FL from FT1, while strains with membrane protein SEQ ID NOs 02 and 40 to 96 produce 3-FL from FT2. CPI data refers to the measurement of 3-FL in the whole broth sample. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point for normalizing all adaptations to this.

[0400] Figure 2: 3-FL output rate expressed as relative percentage (%) obtained in the growth experiment, using strains expressing membrane proteins with SEQ ID NOs 58 to 104 (excluding SEQ ID NO 90) in TU2, SEQ ID NO 90 in TU3, or SEQ ID NOs 02 to 34 in TU10 and expressing α1,3-fucosyltransferase. Strains with membrane proteins having SEQ ID NOs 08 to 30 produce 3-FL from FT1, while strains with membrane proteins having SEQ ID NOs 58 to 104 produce 3-FL from FT2. Strains with membrane protein having SEQ ID NO 02 were tested in combination with FT1 or FT2. The growth experiment was carried out in minimal medium supplemented with 20 g / L lactose as a precursor for 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0401] Figure 3 : Growth rate expressed as relative percentage (%) obtained in the growth experiment, using strains expressing membrane proteins with SEQ ID NOs 08, 14, 18, or 22 in TU10 and expressing α1,3-fucosyltransferase FT1. The growth experiment was carried out in minimal medium supplemented with 20 g / L lactose as a precursor for 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0402] Figure 4 : 3-FL output rate expressed as relative percentage (%) obtained in the growth experiment, using strains expressing membrane protein with SEQ ID NO 28 in TU10 and expressing α1,3-fucosyltransferase FT1. The growth experiment was carried out in minimal medium supplemented with 45 g / L lactose as a precursor for 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0403] Figure 5 : CPI expressed as relative percentage (%) obtained in the growth experiment, using strains expressing membrane protein with SEQ ID NO 28 in TU10 and expressing α1,3-fucosyltransferase FT1. CPI data refers to 3-FL measurements in the whole broth sample. The growth experiment was carried out in minimal medium supplemented with 90 g / L lactose as a precursor for 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0404] Figure 6: 3FL output rate obtained in the growth experiment, expressed as relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 10 or 16 in TU10 and expressing α1,3-fucosyltransferase FT1. The growth experiment was carried out in a minimal medium supplemented with 90 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0405] Figure 7 : CPI obtained in the growth experiment, expressed as relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 10, 16 or 28 in TU10 and expressing α1,3-fucosyltransferase FT1. The growth experiment was carried out in a minimal medium supplemented with 100 g / L sucrose and 90 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0406] Figure 8 : Growth rate obtained in the growth experiment, expressed as relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 28 in TU10 and α1,3-fucosyltransferase FT1. The growth experiment was carried out in a minimal medium supplemented with 100 g / L sucrose and 90 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0407] Figure 9 : CPI obtained in the growth experiment, expressed as relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 22 in TU10 and α1,3-fucosyltransferase FT1. The growth experiment was carried out in a minimal medium supplemented with 5 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0408] Figure 10 : CPI obtained in the growth experiment, expressed as relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 02 or 28 in TU10 from the host genome and expressing α1,3-fucosyltransferase FT1 or FT2 from a plasmid. Thus, the gene having SEQ ID NO 01 was integrated into the EcLdhA locus, and the gene having SEQ ID NO 27 was integrated into the EcSetA locus. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0409] Figure 11: 3-FL output rate obtained in the growth experiment, expressed as relative percentage (%), using a strain expressing a membrane protein with SEQ ID NO 02 or 28 in TU10 from the host genome and expressing α1,3-fucosyltransferase FT2 from a plasmid. Thus, the gene with SEQ ID NO 01 was integrated into the EcLdhA locus, and the gene with SEQ ID NO 27 was integrated into the EcSetA locus. The growth experiment was carried out in minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0410] Figure 12: CPI obtained in the growth experiment, expressed as relative percentage (%), using a strain expressing a membrane protein with SEQ ID NO 02, 06, 10, 16, 22, 28, 32, 34, 36, 38, 40, 42, 44 or 50 from different transcription units (TUs) of a plasmid and expressing α1,3-fucosyltransferase FT2 from a plasmid. The growth experiment was carried out in minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0411] Figure 13 : 3-FL output rate obtained in the growth experiment, expressed as relative percentage (%), using a strain expressing a membrane protein with SEQ ID NO 02, 06, 10, 16, 22, 28, 32, 34, 36, 38, 40, 42, 44 or 50 from different transcription units (TUs) of a plasmid and expressing α1,3-fucosyltransferase FT2 from a plasmid. The growth experiment was carried out in minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0412] Figure 14 : CPI (left panel) and 3-FL output rate (right panel) obtained in the growth experiment, expressed as relative percentage (%), using a strain expressing a membrane protein with SEQ ID NO 40, 42, 46 or 48 (cloned as a single gene in TU10 or cloned into its native transcriptional operon structure containing two membrane protein genes and presented on a plasmid), and expressing α1,3-fucosyltransferase FT2 from a plasmid. The growth experiment was carried out in minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0413] Figure 15: CPI and DiFL output rates of hosts producing 2'FL (Figure A) or DiFL (Figure B) expressed as relative percentages (%) obtained in growth experiments, using strains expressing the membrane protein with SEQ ID NO 28 integrated at the EcSetA locus in the host genome (for the membrane protein with SEQ ID NO 28) or the membrane protein with SEQ ID NO 02 or 06 integrated at the EcLdhA locus in the host genome (for the membrane proteins with SEQ ID NO 02 and 06), and expressing α1,2-fucosyltransferase FT3 from a plasmid. The growth experiments were carried out in minimal medium supplemented with 20 g / L lactose as a precursor for 2'-FL and DiFL. The horizontal dashed line represents the set point to which all adaptations were normalized for this.

[0414] Figure 16 : Productivity parameters improved in the batch and fed-batch phases measured in 8 independent fermentation runs with a 3-FL E. coli production host, which overexpresses the membrane protein with SEQ ID NO 02 from the genome and α1,3-fucosyltransferase FT2 from a plasmid. The horizontal dashed line represents the set point to which all adaptations were normalized for this. A reference fermentation was carried out using the same strain lacking the membrane protein gene overexpression cassette. Fermentation was carried out as described in Example 3. CPI, cell performance index (g 3-FL / g biomass); br, whole broth; sn, supernatant; Qp, specific productivity (g 3-FL / g biomass / h); Qs, specific productivity (g sucrose / g biomass / h); Ys, yield for sucrose (g 3-FL / g sucrose); Yx, biomass yield (g biomass / g sucrose); rate, production rate (g 3-FL / L / h); lac_rate, lactose conversion rate (g lactose consumed / hour).

[0415] Figure 17 : Productivity parameters improved in the batch and fed-batch phases measured in a fermentation run with a 3-FL E. coli production host, which overexpresses the membrane protein with SEQ ID NO 06 from the first plasmid and α1,3-fucosyltransferase FT2 from the second plasmid. The horizontal dashed line represents the set point to which all adaptations were normalized for this. A specific reference fermentation was carried out using the same strain lacking the membrane protein gene. Fermentation was carried out as described in Example 3. CPI, cell performance index (g 3-FL / g biomass); br, whole broth; sn, supernatant; Ys, yield for sucrose (g 3-FL / g sucrose); Yx, biomass yield (g biomass / g sucrose).

[0416] Figure 18: CPI obtained in the growth experiment, expressed as a relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 02, 06, 120, 126, 128, 140, 146 or 150 and expressing α1,3-fucosyltransferase FT1 (for the membrane proteins having SEQ ID NO 02 and SEQ ID NO 06) or FT2 (for the other membrane proteins) from a plasmid. The membrane proteins having SEQ ID NO 02 and SEQ ID NO 06 were cloned into TU10. The membrane protein having SEQ ID NO 126 was cloned into TU2. The membrane proteins having SEQ ID NO 120, 140 and 150 were cloned into TU3. The membrane proteins having SEQ ID NO 128 and SEQ ID NO 146 were cloned into TU2 (version v1) or TU3 (version v2). The CPI data refers to the 3-FL measurement in the whole broth sample. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line indicates the set point for normalizing all adaptations to this.

[0417] Figure 19 : 3-FL output rate obtained in the growth experiment, expressed as a relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 126 and SEQ ID NO 128 (version v1) cloned in TU2, SEQ ID NO 128 (version 2) cloned in TU3 and SEQ ID NO 02 cloned in TU10 and expressing α1,3-fucosyltransferase FT1 (for the strains having the membrane protein with SEQ ID NO 02) or FT2 (for the strains having the membrane proteins with SEQ ID NO 126 and 128) from a plasmid. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line indicates the set point for normalizing all adaptations to this.

[0418] Figure 20 : Growth rate obtained in the growth experiment, expressed as a relative percentage (%), using strains expressing a membrane protein having SEQ ID NO 120 and 140 in TU3 and expressing α1,3-fucosyltransferase FT2 from a plasmid. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line indicates the set point for normalizing all adaptations to this.

[0419] Figure 21 : MATGAT table of Example 20 related to EcMdfA.

[0420] Figure 22: MATGAT table of Example 20 related to EcIceT.

[0421] Figure 23 : CPI expressed as relative percentage (%) obtained in the growth experiment. The strains used express membrane proteins with SEQ ID NO 54 cloned in TU1, SEQ ID NO 62, 66, 70, 76, 84, 92, 96, 104 cloned in TU2, SEQ ID NO 58, 64, 72, 74, 94 cloned in TU3, SEQ ID NO 184, 204, 208 cloned in TU11, and SEQ ID NO 52, 56, 60, 80, 82, 88, 90, 98 cloned in TU12, and express α1,3-fucosyltransferase FT2. The CPI data refers to the 3-FL measurement in the whole broth sample. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 3-FL. The horizontal dashed line represents the set point for normalizing all adaptations to this.

[0422] Figure 24 : CPI expressed as relative percentage (%) obtained in the growth experiment. The strains used express membrane proteins with SEQ ID NO 204 or 214 cloned in TU11 and express α1,2-fucosyltransferase FT4. The CPI data refers to the 2'-FL measurement in the whole broth sample. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 2'-FL. The horizontal dashed line represents the set point for normalizing all adaptations to this.

[0423] Figure 25 : 2'-FL output rate expressed as relative percentage (%) obtained in the growth experiment. The strains used express membrane proteins with SEQ ID NO 206, 208, 214, 216, 218 cloned in TU11 and express α1,2-fucosyltransferase FT4. The growth experiment was carried out in a minimal medium supplemented with 20 g / L lactose as a precursor of 2'-FL. The horizontal dashed line represents the set point for normalizing all adaptations to this. Example

[0424] Example 1: Identification of Membrane Protein Families

[0425] HMM is a probabilistic model called profile hidden Markov models. It represents a set of aligned proteins as a position - specific scoring system. Amino acids are scored at each position in the sequence alignment according to their frequency of occurrence (Eddy, S.R. 1998. Profile hidden Markov models. Bioinformatics. 14:755 - 63). It is clear from the numerous databases that use this method for protein classification that HMM has a wide range of applications. These databases include Pfam, InterPro, SMART, TIGRFAM, PIRSF, PANTHER, SFLD, Superfamily, and Gene3D.

[0426] HMMsearch from the HMMER package 3.2.1 released on June 13, 2019 ( http: / / hmmer.org / ) can use this HMM to search a sequence database for sequence homologs. Sequence databases that can be used include, for example but not limited to: NCBInr Protein Database (NR; https: / / www.ncbi.nlm.nih.gov / protein ), UniProt Knowledgebase (UniProtKB, https: / / www.uniprot.org / help / uniprotkb ), and SWISS - PROT database ( https: / / web.expasy.org / docs / swiss-prot_guideline.html ).

[0427] Based on the eggNOG database 1.0.2 released on November 3, 2017 ( https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6324079 / ; http: / / eggnog.embl.de / # / app / home ), the TCDB database released on June 17, 2019 ( http: / / www.tcdb.org / public / tcdb ), InterPro 75.0 released on July 4, 2019 ( https: / / www.ebi.ac.uk / interpro / ), and the PFAM domains of Pfam 32.0 released in September 2018 ( https: / / pfam.xfam.org / ) are used to classify membrane protein families. The eggNOG database is a public database of orthologous relationships, gene evolutionary history, and functional annotations. The Transporter Classification DataBase (TCDB) is similar to the Enzyme Commission (EC) system used to classify enzymes and contains functional and phylogenetic information. The Pfam and InterPro databases are collections of a large number of protein families. Other protein domains such as SMART ( http: / / smart.embl-heidelberg.de / ), TIGRFAM (https: / / www.jcvi.org / tigrfams )、PIRSF( https: / / proteininformationresource.org / pirwww / dbinfo / pirsf.shtml )、PANTHER( http: / / pantherdb.org / )、SFLD( http: / / sfld.rbvi.ucsf.edu / archive / django / index.html )、Superfamily( http: / / supfam.org / ) and Gene3D( http: / / gene3d.biochem.ucl.ac.uk / Gene3D / )、NCBI Conserved Domains( https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi )。

[0428] The identification of eggNOG families was completed by using the stand-alone version of eggNOG-mapper( https: / / github.com / eggnogdb / eggnog-mapper ) based on eggnogdb 1.0.2 released on November 3, 2017. For each eggNOG family, the HMM can be downloaded from the eggNOG website and used for HMMsearch of protein databases.

[0429] The identification of TCDB families was completed by blasting (blastp) against the TCDB database released on June 17, 2019. New members of the obtained families can be retrieved on the website (http: / / www.tcdb.org / download.php). The Fasta file can be used as the input to the protein database in blastp.

[0430] The identification of PFAM domains was completed by online search on https: / / pfam.xfam.org / search#tabview=tab1 released in September 2018. The HMMs of the obtained families have been downloaded in "Curation&model". Using this model for HMMsearch of protein databases will identify new family members. Sequences containing InterPro hits can also be downloaded from the PFAM website.

[0431] By using either https: / / www.ebi.ac.uk / interpro / or the stand-alone version of InterProScan( https: / / www.ebi.ac.uk / interpro / download.html)Online tools on complete the identification of InterPro (super) families, domains, and sites. InterPro is a composite database that combines information from many databases of protein motifs and domains. HMMs for InterPro domains and / or (super) families can be obtained from InterProScan and can be used to identify new family members in protein databases. Sequences containing InterPro hits can also be downloaded from the InterPro website ("Protein Matched") or can be queried on the UniProt website ( https: / / www.uniprot.org ).

[0432] Example 2: Identification of Membrane Proteins or Protein Sequences Useful in the Methods of the Invention

[0433] The first set of membrane proteins or protein sequences was discovered by identifying PFAM domains of membrane proteins found in the neighborhood of fucosyltransferases and selecting membrane proteins with any of the identified PFAM domains, as illustrated in Example 1. Protein identifiers belonging to the fucosyltransferase families IPR001053 (GT10) and IPR002516 (GT11) were extracted from UniProtKB / trembl, as defined in InterPro 75.0 released on July 4, 2019. These identifiers were used as input to the Genome Neighborhood Tool https: / / efi.igb.illinois.edu / efi-gnt / released on June 19, 2019. EFI-GNT (EFI Genome Neighborhood Tool) allows exploration of genomic neighborhoods and focuses on placing protein families and superfamilies in a genomic context. A Sequence Similarity Network (SSN) was used as input. Each sequence in the SSN was used as a query to query its genomic neighborhood. EFI-GNT is able to explore the genomic neighborhoods of sequences in the SSN clusters to facilitate their functional assignment.

[0434] A neighborhood window size of 14 was selected. Adjacent genes were classified according to their PFAM domains. Membrane proteins with the following PFAM domains were proximal to GT10 (IPR001503) and GT11 (IPR002516) fucosyltransferases: PF00005, PF00006, PF00023, PF00083, PF00092, PF00115, PF00116, PF00122, PF00209, PF00213, PF00230, PF00231, PF00254, PF00359, PF00375, PF00381, PF00391, PF00401, PF00403, PF00474, PF00484, PF00520, PF00528, PF00529, PF00543, PF00571, PF00593, PF00625, PF00654, PF00664, PF00689, PF00690, PF00702, PF00860, PF00873, PF00892, PF00893, PF00902, PF00909, PF00916, PF00939, PF00999, PF01032, PF01061, PF01103, PF01203, PF01235, PF01384, PF01496, PF01544, PF01547, PF01554, PF01566, PF01578, PF01614, PF01618, PF01656, PF01699, PF01740, PF01741, PF01758, PF01810, PF01813, PF01891, PF01895, PF01899, PF01943, PF02026, PF02080, PF02133, PF02136, PF02225, PF02254, PF02277, PF02302, PF02321, PF02355, PF02378, PF02386, PF02417, PF02447, PF02501, PF02563, PF02632, PF02652, PF02653, PF02690, PF02706, PF02874, PF02896, PF03030, PF03083, PF03186, PF03222, PF03412, PF03459, PF03471, PF03544, PF03547, PF03548, PF03567, PF03605, PF03606, PF03610, PF03616, PF03814, PF03840, PF03865, PF03932, PF04193, PF04277, PF04389, PF04966,PF05134, PF05140, PF05524, PF05552, PF05977, PF06251, PF06826, PF06835, PF07264, PF07549, PF07660, PF07670, PF07685, PF07690, PF07715, PF07885, PF07969, PF08239, PF08279, PF08334, PF08352, PF08402, PF08479, PF10531, PF11356, PF11612, PF12156, PF12399, PF12796, PF12822, PF12848, PF12974, PF13306, PF13347, PF13409, PF13410, PF13416, PF13417, PF13440, PF13442, PF13462, PF13466, PF13473, PF13499, PF13505, PF13520, PF13531, PF13599, PF13609, PF13637, PF13807, PF13855, PF14524, PF14667, PF16327, PF17912 and PF18412.,

[0435] Example 3: Materials and Methods for Escherichia coli

[0436] Culture Medium

[0437] Luria Broth (LB) medium consists of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR, Leuven, Belgium). The basal medium used in the culture experiments in 96-well plates or shake flasks contains 2.00 g / L NH4Cl, 5.00 g / L (NH4)2SO4, 2.993 g / L KH2PO4, 7.315 g / L K2HPO4, 8.372 g / L MOPS, 0.5 g / L NaCl, 0.5 g / L MgSO4·7H2O, 30 g / L sucrose or other carbon sources specified in the examples, 1 ml / L vitamin solution, 100 μL / L molybdate solution, and 1 mL / L selenium solution. As specified in the respective examples, 20 or 45 g / L lactose is additionally added to the medium as a precursor. The pH of the medium is set to 7 using 1 M KOH. The vitamin solution consists of 3.6 g / L FeCl2·4H2O, 5 g / L CaCl2·2H2O, 1.3 g / L MnCl2·2H2O, 0.38 g / L CuCl2·2H2O, 0.5 g / L CoCl2·6H2O, 0.94 g / L ZnCl2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA·2H2O, and 1.01 g / L thiamine hydrochloride. The molybdate solution contains 0.967 g / L NaMoO4·2H2O. The selenium solution contains 42 g / L SeO2.

[0438] The basal medium for fermentation contains 6.75 g / L NH4Cl, 1.25 g / L (NH4)2SO4, 2.93 g / L KH2PO4, and 7.31 g / L K2HPO4, 0.5 g / L NaCl, 0.5 g / L MgSO4·7H2O, 30 g / L sucrose, 1 ml / L vitamin solution, 100 μL / L molybdate solution, and 1 mL / L selenium solution, with the same composition as above. As specified in the respective examples, 20 g / L lactose is additionally added to the medium as a precursor.

[0439] The complex medium is sterilized by autoclaving (121 °C, 21'), and the basal medium is sterilized by filtration (0.22 μm Sartorius). When necessary, the medium is made selective by adding antibiotics (such as chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L), and / or kanamycin (50 mg / L)).

[0440] Plasmid

[0441] The plasmids pKD46 (Red helper plasmid, ampicillin resistance), pKD3 (containing the FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (containing the FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expressing FLP recombinase activity) were obtained from Professor R. Cunin (Free University of Brussels, Belgium, 2007).

[0442] Plasmids for membrane proteins and for fucosyltransferase expression were constructed using Golden Gate assembly in pSC101 ori (Rep10-v3) and pMB1 ori containing the backbone vector. All membrane protein and fucosyltransferase coding genes were synthesized at Twist Biosciences (San Francisco, USA). The polynucleotide sequences of the membrane proteins and the corresponding membrane protein polypeptides are listed in Table 1 as shown in SEQ ID NOs 1-196 and SEQ ID NOs 204-218. The fucosyltransferases used in the attached examples are 3-fucosyltransferase FT1 (which has nucleic acid and protein sequences SEQ ID NOs 197 and 198, respectively) and FT2 (having SEQ ID NOs 199 and 200). The 2-fucosyltransferase used is HpFutC having SEQ ID NOs 201 and 202, herein referred to as FT3, and FT4 having nucleic acid and protein sequences SEQ ID NOs 219 and 220, respectively. The membrane protein and fucosyltransferase genes were expressed in different transcription units (TUs) using the specific promoter, UTR, and terminator combinations listed in Table 2. These genes were expressed using the promoters MutalikP5 (“PROM0005_MutalikP5”), MutalikP12 (“PROM0012_MutalikP12”), apFAB146 (“PROM0032”), and MutalikP10 (“PROM0010_MutalikP10”) (as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360)) and two promoters p22 (PROM0015_p22) and p14 (PROM0016_p14) as described by De Mey et al. (BMC Biotechnology 2007, 7:34)). The UTRs used include Gene10-LeuAB-BCD2 (“UTR0002_Gene10-LeuAB-BCD2”), BCD1 (“UTR003_BCD1”), Gene10_LeuL (“UTR0011_Gene10_LeuL”), ThrA_BCD2 (“UTR0013_ThrA_BCD2”), GalE_LeuAB (“UTR0014_GalE_LeuAB”), GalE_lptFG (“UTR0038_GalE_IptFG”), and uspF_iptFG (“UTR0055_uspF_iptFG”) (as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360)).The terminator used in the examples is TER0010_T7 Early as described by Dunn et al. (Nucleic Acids Res. 1980, 8(10), 2119-32). Table 3 shows an overview of the transcription units used in the examples by the combination of the above promoters UTR and terminators. Codon usage can be further promoted by optimizing codon usage for the expression host. Optimize the gene using the vendor's tools.

[0443] Table 1

[0444]

[0445]

[0446]

[0447]

[0448] Table 2

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455] Table 3

[0456]

[0457]

[0458]

[0459] The plasmid was in the host Escherichia coli DH5alpha purchased from Invitrogen (F - ,phi80dlacZdeltaM15,delta(lacZYAargF)U169,deoR,recA1,endA1,hsdR17(rk - ,mk + ),phoA,supE44,lambda -,remain in (thi-1, gyrA96, relA1).

[0460] Strains and Mutations

[0461] Escherichia coli K12 MG1655 [lambda-, F-, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740 in March 2007. Gene disruption and gene introduction were carried out using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640 - 6645). This technique is based on antibiotic selection after homologous recombination by lambda Red recombinase. Subsequent catalysis by the flippase recombinase ensures the removal of the antibiotic selection cassette in the final production strain.

[0462] Transformants carrying the Red helper plasmid pKD46 were grown in 10 ml of LB medium with ampicillin (100 mg / L) and L-arabinose (10 mM) at 30 °C to an OD 600nm of 0.6. The cells were made electrocompetent by washing the cells first with 50 ml of ice-cold water and then with 1 ml of ice-cold water. Then, the cells were resuspended in 50 μl of ice-cold water. Using a Gene Pulser TM (BioRad) (600 Ω, 25 μF D and 250 V), 50 μl of cells and 10 - 100 ng of linear double-stranded DNA product were electroporated.

[0463] After electroporation, the cells were added to 1 ml of LB medium, incubated at 37 °C for 1 h, and finally plated on LB agar containing 25 mg / L chloramphenicol or 50 mg / L kanamycin to select for antibiotic-resistant transformants. The selected mutants were verified by PCR using primers upstream and downstream of the modified region and grown at 42 °C in LB agar to lose the helper plasmid. The mutants were tested for ampicillin sensitivity.

[0464] Linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivatives as templates. A part of the sequence of the primers used was complementary to the template, and the other part was complementary to one side where recombination must occur on the chromosomal DNA. For genome knockout, the regions of homology were designed 50 - nt upstream and 50 - nt downstream of the start codon and stop codon of the target gene. For genome knock-in, the transcription start point (+1) must be respected. The PCR products were PCR purified, digested with DpnI, re-purified from the agarose gel, and suspended in the elution buffer (5 mM Tris, pH 8.0).

[0465] Selected mutants (chloramphenicol or kanamycin resistant) were transformed with the pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and heat induction of FLP synthesis. Ampicillin-resistant transformants were selected at 30 °C, and then some colonies were purified at 42 °C in LB, and then tested for the loss of all antibiotic resistance and FLP helper plasmids. Gene knockout and knock-in were checked using control primers (Fw / Rv-gene-out).

[0466] Mutant strains were generated from Escherichia coli K12 MG1655 by knocking out the genes lacZ, lacY, lacA, glgC, agp, pfkA, pfkB, pgi, arcA, iclR, wcaJ, lon, and thyA. In addition, the Escherichia coli lacY gene, the fructokinase gene (frk) from Zymomonas mobilis, the sucrose transporter of Escherichia coli W (cscB), and the sucrose phosphorylase (SP) from Bifidobacterium adolescentis were knocked into the genome and expressed constitutively. The constitutive promoter was derived from the promoter library described by De Mey et al. (BMC Biotechnology, 2007). These gene modifications are also described in WO2016075243 and WO2012007481. As described in the same document, the α1,3- or α1,2-fucosyltransferase gene was delivered to the mutant strain by plasmid. All membrane protein genes were evaluated in this mutant strain derived from Escherichia coli K12 MG1655. Membrane proteins present on plasmids or integrated into the host genome (at the setA or ldhA loci) were evaluated. All strains were stored in cryotubes at -80 °C (LB cultures were mixed with 70% glycerol in a 1:1 ratio).

[0467] Alternative mutant strains can be derived from Escherichia coli K12 JM109, in which the genes lacZ, rcsA, and wcaJ are knocked out. As described above, the α1,3- or α1,2-fucosyltransferase gene was delivered to the mutant strain by plasmid, resulting in the production of 2'-fucosyllactose, 3-fucosyllactose, or 2',3-difucosyllactose. Membrane protein genes were evaluated in the same manner as described above. The strains were able to internalize lactose via allolactose or IPTG, inducing the lactose permease gene lacY.

[0468] Another alternative mutant strain can be obtained from Escherichia coli BL21. The genes lacZ, fucI, fucK, and wzxC - wcaJ are knocked out in the said strain. To enhance the synthesis of GDP - fucose in the said mutant strain, the genes encoding phosphomannomutase (manB), mannose - 1 - phosphate guanylyltransferase (manC), GDP - mannose - 4,6 - dehydratase (gmd), and GDP - L - fucose synthase (wcaG) from Escherichia coli K12 are overexpressed in a similar manner as above. Intracellular lactose synthesis is accomplished by overexpressing the gene encoding β - 1,4 - galactosyltransferase encoded by the gene lgtB. To enhance the synthesis of UDP - galactose, the operon encoding galETKM is knocked out, and the gene encoding UDP - glucose epimerase is overexpressed. As described above, the α1,3 - or α1,2 - fucosyltransferase gene is delivered to the said mutant strain by plasmid, resulting in the production of 2'-fucosyllactose, 3 - fucosyllactose, or 2',3 - difucosyllactose. The membrane protein genes are evaluated in the same manner as above.

[0469] Another alternative mutant strain can be obtained from Escherichia coli K12. The genes lacZ, fucI, fucK, and wzxC - wcaJ are knocked out in the said strain. To enhance the synthesis of GDP - fucose in the said mutant strain, the genes encoding phosphomannomutase (manB), mannose - 1 - phosphate guanylyltransferase (manC), GDP - mannose - 4,6 - dehydratase (gmd), and GDP - L - fucose synthase (wcaG) from Escherichia coli K12 are overexpressed in a similar manner as above. Additionally, the said strain is modified by genomic knock - in of the fucose permease (fucP) gene from Escherichia coli and the bifunctional fucose kinase / fucose - 1 - phosphate guanylyltransferase (fkp) gene from Bacteroides fragilis. As described above, the α1,3 - or α1,2 - fucosyltransferase gene is delivered to the said mutant strain by plasmid, resulting in the production of 2'-fucosyllactose, 3 - fucosyllactose, or 2',3 - difucosyllactose. The membrane protein genes are evaluated in the same manner as above. The said strain is capable of internalizing lactose through allolactose or IPTG, inducing the lactose permease gene lacY.

[0470] Another alternative mutant strain can be obtained from Escherichia coli K12. The lacZ and wzxC - wcaJ genes are knocked out in the said strain. To improve the synthesis of GDP - fucose in the said mutant strain, the genes encoding phosphomannomutase (manB), mannose - 1 - phosphate guanylyltransferase (manC), GDP - mannose - 4,6 - dehydratase (gmd), and GDP - L - fucose synthase (wcaG) from Escherichia coli K12 are overexpressed in a similar manner as described above. To improve the formation of fructose - 6 - phosphate from gluconeogenic substrates such as glycerol, acetate, lactate, ethanol, succinate, pyruvate, the genes encoding phosphofructokinase (pfkA and pfkB) are knocked out and the genes encoding fructose - 1,6 - bisphosphate aldolase (fbaB) and heterologous fructose - 1,6 - bisphosphatase (fbpase) from Pisum sativum are overexpressed. As described above, the α1,3 - or α1,2 - fucosyltransferase gene is delivered to the said mutant strain by plasmid, resulting in the production of 2'-fucosyllactose, 3 - fucosyllactose, or 2',3 - difucosyllactose. The membrane protein genes are evaluated in the same manner as described above.

[0471] Culture Conditions

[0472] The pre - culture for the 96 - well microtiter plate experiment starts from a cryovial, in 150 μL of LB, and is incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture is used as the inoculum for the 96 - well square microtiter plate and is diluted 400 - fold with 400 μL of minimal medium. Each strain is grown as biological replicates in multiple wells of the 96 - well plate. Then these final 96 - well culture plates are incubated at 37 °C on an orbital shaker at 800 rpm for 72 hours or shorter or longer. At the end of the culture experiment, samples are taken from each well to measure the sugar concentration in the broth supernatant (extracellular sugar concentration, after centrifuging the cells 5 times), or the culture broth is boiled at 90 °C for 15 minutes and then centrifuged to pellet the cells ( = whole broth measurement, the average of intracellular and extracellular sugar concentrations).

[0473] In addition, the culture is diluted to measure the optical density at 600 nm. The cell performance index or CPI is determined as the relative percentage compared to the reference strain by dividing the fucosyllactose concentration measured in the whole broth by the biomass. The biomass is empirically determined to be approximately 1 / 3 of the optical density measured at 600 nm. The fucosyllactose export rate is determined as the relative percentage compared to the reference strain by dividing the fucosyllactose concentration measured in the supernatant by the fucosyllactose concentration measured in the whole broth.

[0474] The pre-culture of the bioreactor starts from a whole 1 mL cryovial of a certain strain, inoculated into 250 mL or 500 mL of basal medium in a 1 L or 2.5 L shake flask, and cultured at 37 °C on an orbital shaker at 200 rpm for 24 hours. Then inoculate a 5 L bioreactor (inoculate 250 mL in 2 L of batch medium); this process is controlled by the MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). The culture conditions are set at 37 °C with maximum agitation; the pressure gas flow rate depends on the strain and the bioreactor. The pH is controlled at 6.8 using 0.5 M H2SO4 and 20% NH4OH. Cool the exhaust gas. Add a 10% silicone antifoam solution when foaming occurs during fermentation.

[0475] Optical Density

[0476] The cell density of the culture was often monitored by measuring the optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or using a Spark 10M microplate reader, Tecan, Switzerland).

[0477] Productivity

[0478] The specific productivity Qp is the specific productivity of the fucosyllactose product, usually expressed as the product mass unit per mass unit of biomass per unit time (= g fucosyllactose / g biomass / h). By measuring the amount of product formed and biomass at the end of each stage and the time frame of each stage, the Qp value for each stage of the fermentation run (i.e., the batch and fed-batch stages) was determined.

[0479] The specific productivity Qs is the specific consumption rate of a substrate, such as sucrose, usually expressed as the substrate mass unit per mass unit of biomass per unit time (= g sucrose / g biomass / h). By measuring the total amount of sucrose consumed and the biomass formed at the end of each stage and the time frame of each stage, the Qs value has been determined for each stage of the fermentation run (i.e., the batch and fed-batch stages).

[0480] The yield Ys of sucrose is the fraction of the product made from the substrate, usually expressed as the product mass unit per mass unit of substrate (= g fucosyllactose / g sucrose). By measuring the total amount of fucosyllactose produced and the total amount of sucrose consumed at the end of each stage, the Ys for each stage of the fermentation run, i.e., the batch and fed-batch stages, has been determined.

[0481] The biomass productivity Yx is the fraction of biomass made from the substrate, usually expressed as mass units of biomass per mass unit of substrate (=g biomass / g sucrose). For each stage of the fermentation run, namely the batch and fed-batch stages, Yp has been determined by measuring the total amount of biomass produced and the total amount of sucrose consumed at the end of each stage.

[0482] The rate is the speed at which the product is produced during the fermentation run, usually expressed as the concentration of the product produced per unit time (=g fucosyllactose / L / h). This rate is determined by measuring the concentration of fucosyllactose produced at the end of the fed-batch stage and dividing this concentration by the total fermentation time.

[0483] The lactose conversion rate is the speed at which lactose is consumed during the fermentation run, usually expressed as mass units of lactose per unit time (=g lactose consumed / h). The lactose conversion rate is determined by measuring the total lactose consumed during the fermentation run divided by the total fermentation time.

[0484] Growth Rate / Velocity Measurement

[0485] The maximum growth rate (μMax) was calculated using the R package grofit based on the optical density observed at 600 nm.

[0486] Liquid Chromatography

[0487] Standards of 2'-fucosyllactose, 3-fucosyllactose, and 2',3-difucosyllactose were synthesized in-house. Other standards such as but not limited to lactose, sucrose, glucose, glycerol, fructose were purchased from Sigma. Carbohydrates were analyzed by the HPLC-RI (Waters, USA) method, where RI (refractive index) detects the change in refractive index of the mobile phase containing the sample. Sugars were separated in a constant flow using an X-Bridge column (Waters X-bridge HPLC column, USA) and a mobile phase containing 75 ml of acetonitrile, 25 ml of ultrapure water, and 0.15 ml of triethylamine. The column size was 4.6 x 150 mm and the particle size was 3.5 μm. The column temperature was set at 35 °C and the pump flow rate was 1 mL / min.

[0488] Normalization of Data

[0489] For all types of culture conditions, the data obtained from the mutant strains were normalized against the data obtained from the reference strain under the same culture conditions, where the reference strain has the same genetic background as the mutant strain but lacks the membrane protein expression cassette. The horizontal dashed line on each graph shown in the examples represents the set point to which all adaptations were normalized. All data are given as relative percentages against this set point.

[0490] Example 4: Identification of membrane proteins that enhance the production of 3-fucosyllactose (3-FL) in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 20 g / L lactose Figure 1

[0491] Experiments were established to evaluate the ability of membrane proteins to enhance the production of fucosyllactose in host cells growing in minimal medium supplemented with 20 g / L lactose. Membrane proteins having SEQ ID NOs 02, 04, 06, 18, 20, 22, 26, 28, 30, 32, 34, 40, 42, 44, 58, 62, 64, 66, 70, 72, 74, 82, 84, 90, 92, 94, and 96 were shown to be able to enhance 3-FL production in 3-FL-producing hosts expressing α1,3-fucosyltransferase FT1 or FT2. The candidate genes were combined in transcription units TU2, TU3, or TU10 and presented to the 3-FL-producing host on a pSC101 plasmid. Growth experiments were performed according to the culture conditions provided in Example 3. Example 5: Identification of membrane proteins that enhance the secretion of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 20 g / L lactose Shows the CPI of the strain expressed as a relative percentage compared to the corresponding reference strain.

[0492] Figure 2 Example 6: Identification of membrane proteins that enhance the growth rate in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 20 g / L lactose

[0493] Experiments were established to evaluate the ability of membrane proteins to enhance the secretion of fucosyllactose in host cells growing in minimal medium supplemented with 20 g / L lactose. Membrane proteins having SEQ ID NOs 02, 08, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 34, 58, 62, 64, 66, 70, 72, 74, 76, 82, 84, 90, 92, 94, 96, and 104 were shown to be able to enhance the secretion of 3-FL that is produced intracellularly in a 3-FL bacterial production host expressing α1,3-fucosyltransferase FT1 or FT2. The candidate genes were combined in transcription units TU2, TU3, or TU10 and presented to the 3-FL-producing host on a pSC101 plasmid. Growth experiments were performed according to the culture conditions provided in Example 3. Figure 3 Shows the output rate of 3-FL in the strain expressed as a relative percentage compared to the corresponding reference strain.

[0494] Example 7: Identification of membrane proteins that enhance the secretion of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 45 g / L lactose Figure 4

[0495] Experiments were established to evaluate the ability of membrane proteins to affect the growth rate of host cells in minimal medium supplemented with 20 g / L lactose. Membrane proteins with SEQ ID NO 08, 14, 18, and 22 were shown to be able to enhance the growth rate of 3-FL producing hosts expressing α1,3-fucosyltransferase FT1 or FT2. The candidate genes were combined in transcription units TU2, TU3, or TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were carried out according to the culture conditions provided in Example 3. Example 8: Identification of membrane proteins that enhance the production of 3-FL in Escherichia coli hosts cultured for 72 The growth rate of the strain, expressed as a relative percentage compared to the corresponding reference strain, is shown.

[0496] h in a growth experiment in minimal medium supplemented with 90 g / L lactose

[0497] Experiments were established to evaluate the effectiveness of a series of identified membrane proteins in enhancing fucosyllactose secretion of host cells growing in minimal medium supplemented with 45 g / L lactose. The membrane protein with SEQ ID NO 28 was shown to be able to enhance 3-FL secretion in a 3-FL producing host expressing α1,3-fucosyltransferase FT1. The gene with SEQ ID NO 27 was combined in transcription unit TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were carried out using minimal medium supplemented with 45 g / L lactose according to the culture conditions provided in Example 3. Figure 5 The CPI of the strain, expressed as a relative percentage compared to the corresponding reference strain, is shown.

[0498] Example 9: Identification of membrane proteins that increase the secretion of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 90 g / L lactose Figure 6 Example 10: Identification of membrane proteins that increase the yield of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 100 g / L sucrose and 90 g / L lactose Figure 7

[0499] Experiments were established to evaluate the ability of a series of membrane proteins to enhance fucosyllactose production in minimal medium supplemented with 90 g / L lactose. The membrane protein with SEQ ID NO 28 was shown to be able to enhance 3-FL production in a 3-FL producing host expressing α1,3-fucosyltransferase FT1. The gene with SEQ ID NO 27 was combined in transcription unit TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were carried out using minimal medium supplemented with 90 g / L lactose according to the culture conditions provided in Example 3. Example 11: Identification of membrane proteins that increase the growth rate of Escherichia coli hosts when cultured for 72 h in a growth experiment in minimal medium supplemented with 100 g / L sucrose and 90 g / L lactose The CPI, expressed as a relative percentage compared to the corresponding reference strain, is shown.

[0500] Figure 8 Example 12: Identification of membrane proteins that increase the production of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 5 g / L lactose Figure 9

[0501] Experiments were established to evaluate the ability of a series of membrane proteins to increase the secretion of fucosyllactose by host cells growing in minimal medium supplemented with 90 g / L lactose. Membrane proteins with SEQ ID NOs 10 and 16 were shown to be able to enhance 3-FL secretion in a 3-FL producing host expressing α1,3-fucosyltransferase FT1. The candidate genes were combined in transcription unit TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were performed using minimal medium supplemented with 90 g / L lactose according to the culture conditions provided in Example 3. Example 13: Identification of membrane proteins that increase the production and / or secretion of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 20 g / L lactose when integrated into the host genome Shows the output rate of 3-FL expressed as a relative percentage compared to the corresponding reference strain.

[0502] Figure 10 Figure 11

[0503] Experiments were established to evaluate the ability of a series of membrane proteins to increase the production of fucosyllactose in host cells growing in minimal medium supplemented with 100 g / L sucrose and 90 / L lactose. Membrane proteins with SEQ ID NOs 10, 16, and 28 were shown to be able to enhance 3-FL production in a 3-FL producing host expressing α1,3-fucosyltransferase FT1. All candidate genes were combined in transcription unit TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were performed using minimal medium supplemented with 100 g / L sucrose and 90 g / L lactose according to the culture conditions provided in Example 3. Example 14: Membrane proteins independent of the transcriptional units into which they are cloned enhance 3-FL production Shows the CPI expressed as a relative percentage compared to the corresponding reference strain.

[0504] and / or 3-FL secretion in Escherichia coli hosts Figure 13

[0505] Experiments were established to evaluate the ability of a membrane protein to affect the growth rate of host cells growing in minimal medium supplemented with 100 g / L sucrose and 90 / L lactose. The membrane protein with SEQ ID NO 28 was shown to be able to enhance the growth rate of a 3-FL producing host expressing α1,3-fucosyltransferase FT1. The gene with SEQ ID NO 27 was combined in transcription unit TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were performed using minimal medium supplemented with 100 g / L sucrose and 90 g / L lactose according to the culture conditions provided in Example 3. Example 15: Identification of membrane proteins that enhance the production and / or secretion of 3-FL in Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 20 g / L lactose when cloned in their native transcriptional operon structure Shows the growth rate expressed as a relative percentage compared to the corresponding reference strain.

[0506] Figure 14 Example 16: Identification of membrane proteins that enhance the production and / or secretion of 2'-

[0507] Experiments were established to evaluate the ability of membrane proteins to affect the production of fucosyllactose by host cells growing in minimal medium supplemented with 5 g / L lactose. The membrane protein with SEQ ID NO 22 was shown to be able to enhance 3-FL production in a 3-FL producing host expressing α1,3-fucosyltransferase FT1. The gene with SEQ ID NO 21 was combined in transcription unit TU10 and presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were performed using minimal medium supplemented with 5 g / L lactose according to the culture conditions provided in Example 3. FL and / or DiF and / or the secretion of DiFL in 2'- Shows the CPI expressed as a relative percentage compared to the corresponding reference strain.

[0508] FL Escherichia coli hosts cultured for 72 h in a growth experiment in minimal medium supplemented with 20 g / L lactose Figure 15

[0509] Another series of experiments were established to evaluate the ability of membrane proteins integrated in the genome to increase the production and / or secretion of fucosyllactose in / by host cells cultured in minimal medium supplemented with 20 g / L lactose for 72 h. The membrane proteins with SEQ ID NO 02 and 28 were shown to be able to enhance 3-FL production and / or 3-FL secretion, which is produced intracellularly in a 3-FL producing host expressing α1,3-fucosyltransferase FT1 or FT2. The genes with SEQ ID NO 01 and 27 were combined in transcription unit TU10 and presented as genomic KI at the EcLdhA or EcSetA locus, respectively, to the genome of the 3-FL producing host. Growth experiments were performed according to the culture conditions provided in Example 3. Example 17: Membrane protein MdfA enhances the productivity of 3-FL-producing Escherichia coli hosts in a (30 L) fermentation run Shows the CPI while Figure 16 shows the 3-FL output, both expressed as a relative percentage compared to the corresponding reference strain.

[0510] Example 18: Membrane protein IceT enhances the productivity of 3-FL-producing Escherichia coli hosts in a (30 L) fermentation run Figure 17

[0511] Another series of experiments were established to evaluate the ability of membrane proteins to affect the production and / or secretion of fucosyllactose by host cells. There were several transcription units for cloning in this example. The membrane proteins with SEQ ID NO 02, 06, 10, 16, 22, 28, 32, 34, 36, 38, 40, 42, 44 and 50 were shown to be able to enhance 3-FL production and / or secretion of 3-FL, which is produced intracellularly by α1,3-fucosyltransferase FT2 in a 3-FL producing host. The output genes were cloned into different transcription units and presented to the 3-FL producing host as a cloning vector (pSC101 ori). Growth experiments were performed according to the culture conditions provided in Example 3. Figure 12 shows the CPI while Example 19: Membrane proteins homologous to EcMdfA or EcIceT enhance in minimal medium supplemented with 20 g / L lactoseShows 3-FL outputs, both expressed as relative percentages compared to the respective reference strains.

[0512] ​ ​

[0513] Experiments were established to evaluate the ability of membrane proteins to enhance the production and / or secretion of fucosyllactose in host cells cultured for 72 h in minimal medium supplemented with 20 g / L lactose. This time, the membrane proteins were cloned into their native transcriptional operon structures. Membrane proteins with SEQ ID NOs 40, 42, 46, and 48 were shown to be able to enhance 3-FL production and / or 3-FL secretion in a 3-FL producing host expressing α1,3-fucosyltransferase FT2. All candidate output genes were cloned as single genes in TU10 or cloned in their native transcriptional operon structures containing 2 membrane proteins and presented to the 3-FL producing host on the pSC101 plasmid. Growth experiments were carried out according to the culture conditions provided in Example 3. ​ Shows CPI (left panel) and 3-FL output rate (right panel) expressed as relative percentages compared to the respective reference strains.

[0514] ​ ​

[0515] Experiments were established to evaluate the ability of a series of membrane proteins to enhance the production and / or secretion of fucosyllactose in host cells cultured for 72 h in minimal medium supplemented with 20 g / L lactose. Membrane proteins for 2'-FL and / or diFL production were tested this time. Membrane proteins with SEQ ID NOs 02, 06, and 28 were shown to be able to enhance the production and / or secretion of 2'FL and / or DiFL in a 2'-FL producing host expressing α1,2-fucosyltransferase FT3. The candidate genes were cloned in TU10 and presented on the genome of the 2'-FL producing host using the SetA locus (for the gene with SEQ ID NO 27) or the ldhA locus (for the genes with SEQ ID NOs 01 and 05). Growth experiments were carried out according to the culture conditions provided in Example 3. ​ Shows CPI and DiFL output rate (Figure C) of hosts with 2'FL (Figure A) or DiFL (Figure B) production, each expressed as relative percentages compared to the respective reference strains.

[0516] ​

[0517] The productivity of a 3-FL producing E. coli host expressing a membrane protein gene with SEQ ID NO 01 in TU1 and presented in the host genome at the EcldhA locus and expressing α1,3-fucosyltransferase FT2 from a plasmid was evaluated in a bioreactor setup. Eight fermentation runs were carried out according to the conditions provided in Example 3. Additionally, a reference strain identical to the 3-FL producing host but lacking the membrane protein gene was analyzed in the same fermentation setup. ​ It is shown that, relative to this reference strain, the productivity of the strain overexpressing the membrane protein EcMdfA with SEQ ID NO 02 was increased in eight different fermentation runs.

[0518] ​

[0519] The productivity of another 3-FL producing E. coli host expressing a membrane protein was evaluated in a 30 L bioreactor. The 3-FL strain had a membrane protein gene EcIceT with SEQ ID NO 05 expressed in TU3 from a first plasmid and α1,3-fucosyltransferase FT2 expressed from a second plasmid. A specific reference strain identical to the 3-FL producing host but lacking the membrane protein gene construct was used to analyze the 3-FL productivity under the same fermentation setup. ​ It is shown that, relative to the specific reference strain, the productivity of the strain overexpressing the membrane protein was increased.

[0520] ​ 3-FL production and / or 3-FL secretion and / or growth rate of 3-FL Escherichia coli host cultured for 72 h in growth experiments in the medium

[0521] Experiments were established to evaluate whether a membrane protein with SEQ ID NO 120 (homologous to EclceT with SEQ ID NO 06) and with SEQ ID NO 126, 128, 140, 146, and 150 (homologous to the membrane protein EcMdfA with SEQ ID NO 02) could enhance 3-FL production and / or secretion of 3-FL, which was produced intracellularly by α1,3-fucosyltransferase FT1 or FT2 in a 3-FL producing host, and / or improve the growth rate of the 3-FL E. coli host. The output genes were cloned into different transcription units and presented as a cloning vector (pSC101 ori) to the 3-FL producing host. Growth experiments were carried out according to the culture conditions provided in Example 3. Figure 18 CPI is shown, while Figure 19 3-FL output is shown, Figure 20 and strains with an improved growth rate are shown, each expressed as a relative percentage compared to their respective reference strains.

[0522] Example 20: Calculate the overall percentage identity between polypeptide sequences

[0523] Alignment methods for sequences used in comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e., spanning the entire sequence) alignment of two sequences to maximize the number of matches and minimize the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215:403-10) calculates the percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) in the United States. For example, homologs can be easily identified using the ClustalW multiple sequence alignment algorithm (version 1.83) with default pairwise alignment parameters and the percentage scoring method. One of the methods available in the MatGAT software package can also be used to determine the global percentage of similarity and identity (Campanella et al., BMC Bioinformatics, 2003 Jul 10; 4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences). As will be obvious to those skilled in the art, minor manual editing can be performed to optimize the alignment between conserved motifs. In addition, specific domains can be used instead of the full-length sequence to identify homologs. The sequence identity value can be determined over the entire nucleic acid or amino acid sequence or over the selected domain or conserved motif using the above programs with default parameters. For local alignment, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).

[0524] The MatGAT (Matrix Global Alignment Tool) software was used to determine the global percentage of similarity and identity between the full-length polypeptide sequences for carrying out the method of the present invention (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences. Campanella JJ, Bitincka L, Smalley J; software supervised by Ledion Bitincka). MatGAT generates a similarity / identity matrix of DNA or protein sequences without pre-aligning the data. The program uses the Myers and Miller global alignment algorithm to perform a series of pairwise alignments, calculate similarity and identity, and then put the results into a distance matrix. 1. CYP704-like polypeptide.

[0525] Figure 21 and Figure 22 show exemplary analysis results of the global identity of the full-length polypeptide sequences related to EcMdfA (SEQ ID NO 2) and EcIceT (SEQ ID NO 6). The sequence identity is shown in the upper half of the diagonal dividing line. The parameters used in the comparison were: scoring matrix: Blosum62, first gap: 12, extended gap: 2. The sequence identity (percentage) between the EcMdfA membrane protein and its homologues useful in carrying out the method of the present invention is generally higher than 80%. The sequence identity percentage between the EcIceT membrane protein and its homologues useful in carrying out the method of the present invention is generally higher than 80%.

[0526] Example 21: Identify the membrane proteins that enhance the production of 3-fucosyllactose (3-FL) in Escherichia coli hosts cultured for 72 h in growth experiments in minimal medium supplemented with 20 g / L lactose Figure 23

[0527] Experiments were established to evaluate the ability of membrane proteins to enhance the production of fucosyllactose by host cells growing in minimal medium supplemented with 20 g / L lactose. Membrane proteins having SEQ ID NOs 52, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76, 80, 82, 84, 88, 90, 92, 94, 96, 98, 104, 184, 204 and 208 showed that they were able to enhance 3-FL production in a 3-FL producing host expressing the α1,3-fucosyltransferase FT2. The candidate gene combinations were in transcription units TU1, TU2, TU3, TU11 or TU12 and were presented to the 3-FL producing host on a pSC101 plasmid. Growth experiments were carried out according to the culture conditions provided in Example 3. Example 22: Identify the membrane proteins that enhance the production of 2'-fucosyllactose (2'-FL) in Escherichia coli hosts cultured for 72 h in growth experiments in minimal medium supplemented with 20 g / L lactoseShows the CPI of the strains, expressed as relative percentages compared to their respective reference strains.

[0528] Figure 24 Example 23: Identify the membrane proteins that enhance the secretion of 2'-FL in Escherichia coli hosts cultured for 72 h in growth experiments in minimal medium supplemented with 20 g / L lactose

[0529] Experiments were established to evaluate the ability of membrane proteins to enhance the production of fucosyllactose in host cells growing in minimal medium supplemented with 20 g / L lactose. Membrane proteins having SEQ ID NOs 204 and 214 showed their ability to enhance 2'-FL production in a 2'-FL producing host expressing α1,2-fucosyltransferase FT4. The candidate genes were combined in transcription unit TU11 and presented to the 2'-FL producing host on a pSC101 plasmid. Growth experiments were conducted according to the culture conditions provided in Example 3. Figure 25 Shows the CPI of the strains, expressed as relative percentages compared to their respective reference strains.

[0530] ​ ​

[0531] Experiments were established to evaluate the ability of membrane proteins to enhance the secretion of fucosyllactose in host cells growing in minimal medium supplemented with 20 g / L lactose. Membrane proteins having SEQ ID NOs 206, 208, 214, 216 and 218 showed their ability to enhance the secretion of 2'-FL produced intracellularly in a 2'-FL bacterial producing host expressing α1,2-fucosyltransferase FT4. The candidate genes were combined in transcription unit TU11 and presented to the 2'-FL producing host on a pSC101 plasmid. Growth experiments were conducted according to the culture conditions provided in Example 3. ​ Shows the output rate of 2'-FL in the strains, expressed as relative percentages compared to the corresponding reference strains.

Claims

1. A method for producing fucosyllactose by genetically modified cells, comprising the following steps: - Providing cells capable of producing fucosyllactose, said cells comprising at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a guanosine diphosphate fucose, i.e., GDP-fucose donor, to a lactose acceptor, thereby synthesizing fucosyllactose. - Said cells further comprise: i) a modified expression of an endogenous membrane protein that enables and / or enhances the transport of fucosyllactose and / or ii) an expression of a heterologous membrane protein that enables and / or enhances the transport of fucosyllactose; - Culturing said cells in a culture medium under conditions allowing the production of the desired fucosyllactose, and / or - Isolating fucosyllactose from the culture; Wherein said membrane protein is a transporter selected from the following that enables and / or enhances the transport of fucosyllactose: - MdfA from Escherichia coli K12 MG1655 consisting of SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 consisting of SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 consisting of SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 consisting of SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 consisting of SEQ ID NO 10, YbdA from Escherichia coli K12 MG1655 consisting of SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 consisting of SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 consisting of SEQ ID NO 18, YebQ from Escherichia coli K12 MG1655 consisting of SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 consisting of SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 consisting of SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 consisting of SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 consisting of SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 consisting of SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. Infantis consisting of SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. Infantis consisting of SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. Infantis consisting of SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. Infantis consisting of SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. Infantis consisting of SEQ ID NO 50, CDT2 from Neurospora crassa OR74A consisting of SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 consisting of SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 consisting of SEQ ID NO 58, Eubacterium sp. from Eubacterium sp. consisting of SEQ ID NO 60)The Wzx of CAG:581, the Wzx from Dyadobacter soli consisting of SEQ ID NO 62, the Wzx from Lactococcus raffinolactis consisting of SEQ ID NO 64, the Wzx from Prevotella ruminicola consisting of SEQ ID NO 66, the NAm from Actinobaculum suis consisting of SEQ ID NO 98, the Nap from Niabella drilacis consisting of SEQ ID NO 104, the mdtD from Klebsiella pneumoniae 30684 / NJST258_2 consisting of SEQ ID NO 120, the Cmr from Yokenella regensburgei consisting of SEQ ID NO 126, the MdfA from Cronobacter muytjensii consisting of SEQ ID NO 128, the Cmr from Citrobacter youngae consisting of SEQ ID NO 140, the MdfA from Enterobacter sp. consisting of SEQ ID NO 146, the MdfA from Enterobacter ludwigii EcWSU1 consisting of SEQ ID NO 150, the arabinose efflux protein from Azospirillum brasiliense LMG 04375 consisting of SEQ ID NO 184.

2. The method for producing fucosyllactose according to claim 1, wherein said membrane protein is a transport protein involved in the outer membrane transport of a compound across the cell wall.

3. The method for producing fucosyllactose according to claim 1, said method further comprising at least one of the following steps: i) Lactose feed is added to the culture medium in a continuous manner, with at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, and / or at least 150 grams of lactose per liter of the initial reactor volume, where the reactor volume ranges from 250 mL to 10000 m 3 , and / or such that the final volume of the culture medium is no more than three times, no more than two times, and / or less than 2 times the volume of the culture medium before the addition of the lactose feed; ii) Adding a lactose feed continuously to the culture medium by a feed solution over a period of 1 day, 2 days, 3 days, 4 days, or 5 days; iii) Adding a lactose feed continuously to the culture medium by a lactose feed solution over a period of 1 day, 2 days, 3 days, 4 days, or 5 days, and wherein the concentration of said lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L, or 600 g / L; and / or wherein the pH value of said lactose feed solution is set to 3 to 7, and / or wherein the temperature of said lactose feed solution is maintained at 20°C to 80°C; The method produces a fucosyllactose concentration of at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L, and / or at least 200 g / L in the final volume of the culture medium.

4. The method according to claim 1, wherein the method produces a mixture of fucosyllactose.

5. The method according to claim 1, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose, and / or difucosyllactose.

6. The method according to any one of claims 1-5, wherein the genetically modified cells are selected from the group consisting of microbial cells, plant cells other than plant cells capable of developing into a complete plant, or animal cells other than animal cells capable of developing into a complete animal.

7. The method according to claim 6, wherein the genetically modified cells are Escherichia coli cells.

8. A host cell genetically modified for the production of fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor to synthesize fucosyllactose, the cell further comprising: i) a modified expression of an endogenous membrane protein that enables and / or enhances the transport of fucosyllactose and / or ii) an expression of a heterologous membrane protein that enables and / or enhances the transport of fucosyllactose, and wherein said membrane protein is a transporter membrane protein capable of effecting and / or enhancing the transport of fucosyllactose selected from the group consisting of: MdfA from Escherichia coli K12 MG1655 consisting of SEQ ID NO 02, IceT from Escherichia coli K12 MG1655 consisting of SEQ ID NO 06, YnfM from Escherichia coli K12 MG1655 consisting of SEQ ID NO 04, Yhhs from Escherichia coli K12 MG1655 consisting of SEQ ID NO 08, EmrD from Escherichia coli K12 MG1655 consisting of SEQ ID NO 10, YbdA from Escherichia coli K12 MG1655 consisting of SEQ ID NO 14, YdeE from Escherichia coli K12 MG1655 consisting of SEQ ID NO 16, MhpT from Escherichia coli K12 MG1655 consisting of SEQ ID NO 18, YebQ from Escherichia coli K12 MG1655 consisting of SEQ ID NO 20, YjhB from Escherichia coli K12 MG1655 consisting of SEQ ID NO 22, Bcr from Escherichia coli K12 MG1655 consisting of SEQ ID NO 24, FucP from Escherichia coli K12 MG1655 consisting of SEQ ID NO 26, WzxE from Escherichia coli K12 MG1655 consisting of SEQ ID NO 32, EmrE from Escherichia coli K12 MG1655 consisting of SEQ ID NO 38, Blon_2331 from Bifidobacterium longum subsp. infantis consisting of SEQ ID NO 40, Blon_2332 from Bifidobacterium longum subsp. infantis consisting of SEQ ID NO 42, Blon_0247 from Bifidobacterium longum subsp. infantis consisting of SEQ ID NO 46, Blon_0245 from Bifidobacterium longum subsp. infantis consisting of SEQ ID NO 48, Blon_0345 from Bifidobacterium longum subsp. infantis consisting of SEQ ID NO 50, CDT2 from Neurospora crassa OR74A consisting of SEQ ID NO 52, CDT2 from Aspergillus oryzae RIB40 consisting of SEQ ID NO 54, Wzx from Chitinophaga sp. CF118 consisting of SEQ ID NO 58, Wzx from Eubacterium sp. CAG:581 consisting of SEQ ID NO 60, Wzx from Agrobacterium tumefaciens consisting of SEQ ID NO 62, Wzx from Lactococcus raffinolactis consisting of SEQ ID NO 64, Wzx from Prevotella ruminicola consisting of SEQ ID NO 66NAm from Actinobacillus suis consisting of SEQ ID NO 98, Nap from Niabelladrilacis consisting of SEQ ID NO 104, mdtD from Klebsiella pneumoniae 30684 / NJST258_2 consisting of SEQ ID NO 120, Cmr from Prevotella ruminicola consisting of SEQ ID NO 126, MdfA from Cronobacter muytjensii consisting of SEQ ID NO 128, Cmr from Citrobacter youngae consisting of SEQ ID NO 140, MdfA from Enterobacter sp. consisting of SEQ ID NO 146, MdfA from Enterobacter ludwigii EcWSU1 consisting of SEQ ID NO 150, arabinose efflux protein from Azospirillum brasilense LMG 04375 consisting of SEQ ID NO 184., 9. The host cell according to claim 8, wherein the membrane protein is a transporter protein involved in the outer membrane transport of a compound across the cell wall.

10. The host cell according to claim 8, wherein the cell is selected from the group consisting of microbial cells, plant cells other than plant cells capable of developing into a complete plant, or animal cells other than animal cells capable of developing into a complete animal.

11. The host cell according to claim 8, wherein the cell is an Escherichia coli cell.

12. The host cell according to any one of claims 8 to 11, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose.

13. Use of the host cell according to any one of claims 8 to 12 for the production of fucosyllactose, wherein the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose.

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