NZ793011A - Improved process for the production of fucosylated oligosaccharides - Google Patents

Abstract

The present invention relates to a method for producing fucosylated oligosaccharides by using a recombinant prokaryotic host cell that is cultivated on a gluconeogenic substrate, as well as to the host cell and its use. The host cell is genetically modified in that the activity of a fructose-6-phosphate converting enzyme is abolished or lowered, and the transport of the produced fucosylated oligosaccharide through the cell membrane is facilitated by an exogenous transport protein. hate converting enzyme is abolished or lowered, and the transport of the produced fucosylated oligosaccharide through the cell membrane is facilitated by an exogenous transport protein.

Description

The present invention relates to a method for producing fucosylated accharides by using a recombinant prokaryotic host cell that is cultivated on a gluconeogenic substrate, as well as to the host cell and its use. The host cell is genetically modified in that the activity of a fructose phosphate converting enzyme is abolished or lowered, and the transport of the produced fucosylated oligosaccharide through the cell membrane is facilitated by an exogenous ort protein.

NZ 793011 Improved process for the production of fucosylated oligosaccharides The present application is a divisional of New Zealand patent ation 752719, which is the national phase entry of PCT international application shed as incorporated by reference herein. [0001A] The present ion relates to a method for the production of fucosylated oligosaccharides using a genetically ed prokaryotic host cell, as well as to a host cell employed in this method and its use for producing fucosylated oligosaccharides to high .

OUND OF THE INVENTION Human milk ents a complex mixture of carbohydrates, fats, ns , vitamins, minerals and trace elements. The by far most predominant fraction is represented by carbohydrates, which can be further divided into lactose and more complex oligosaccharides (Human milk oligosaccharides, HMO). Whereas e is used as an energy source, the complex oligosaccharides are not metabolized by the infant. The fraction of complex oligosaccharides accounts for up to 20% of the total carbohydrate fraction and consists of more than 200 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and thus cannot be found in large quantities in the milk of other mammals. imately 200 urally-diverse HMOs have been identified so far and numerous beneficial properties have been reported. HMOs are not digested by breastfed infants, but represent a valuable carbon and energy source for beneficial ia of the genera Bifidobacteria, Lactobacillus and Bacteroides in the intestine, causing them to become dominant in the gut and allowing them to outcompete pathogens, thus preventing infections of the gut epithelium. However, HMOs also bind directly to pathogenic bacteria, protozoa and viruses, blocking pathogen–host interactions by mimicking glycan cell surface receptors and thereby protecting the breastfed child from infectious diseases.

The most prominent oligosaccharide is osyllactose. Further promi- nent HMOs present in human milk are 3—fucosyllactose, lacto-N-tetrao‘se, lacto-N- neotetraose and the lacto-N—fucopentaoses. Besides these neutral oligosaccharides, also acidic HMOs can be found in human milk, like for e.g. 3'-sialyllactose, 6’-sialyllactose and sialyllacto-N-tetraose a, b, and c, or sialyllacto-N—fucpentaose || ect. These structures are closely related to epitopes of epithelial cell e glycoconjugates, the Lewis lood group antigens, and the structural homology of HMO to epithelial epitopes accounts for protective ties against bacterial pathogens.

Due to their beneficial ties, HMOs are favored for inclusion as in- gredients in infant formulae and other food products, itating the production of HMOs in large quantities, up to the multi—ton scale.

Due to the limited supply and difficulties of ing pure fractions of individual human milk oligosaccharides, chemical routes to some of these complex molecules were ped. However, chemical and biocatalytic approaches proved to be not commercially sustainable, and, furthermore, particularly chemical synthetic routes to human milk oligosaccharides involve several noxious chemicals, which impose the risk to contaminate the final product.

Due to the challenges involved in the chemical synthesis of human milk oligosaccharides, several enzymatic methods and fermentative approaches were developed.

Today, for l HMOs such as 2'-fucosyllactose, 3-fucosyllactose, lacto—N— tetraose, N-neotetraose, lacto-N-fucopentaose l, lacto-N-difucohexaose ll, 3’— sialyllactose and 6’-sialyllactose fermentative approaches have been developed, using mainly genetically engineered bacterial strains such as recombinant Escherichia coli.

However, even the most efficient processes based on bacterial fermen- tation, which are ble today, do not or hardly achieve HMO titers greater than 20 g/L in the culture broth. Industrial-scale ses must typically exceed titers of 50 g/L although 100 g/L is more desirable.

Thus, it is an object of the present invention to provide for an improved fermentation process by means of which a biosynthesis of fucosylated oligosaccharides, in particular of 2'-fucosyllactose, is d at a titer exceeding more than 100 g/L.

Y OF THE INVENTION This, and other objects are solved by a method for the production of fu- cosylated accharides using a genetically modified prokaryotic host cell, the method comprising the steps of: - providing a host cell, which has been genetically modified, such, that at least (i) the ty of a fructosephosphate-converting enzyme, which in the unmodified host cell has a regular level, is lowered or abolished; (ii) at least one gene encoding an enzyme necessary for the de novo sis of GDP-fucose is overexpressed in the host cell; (iii) an exogenous gene, encoding a fucosyltrans- ferase, preferably an alpha-1,2—fucosyltransferase and/or alpha-1,3- fucosyltransferase, is expressed, preferably overexpressed, in the host cell; - cultivating said cally modified host cell in a cultivation medium containing a carbon and energy source that is ed from at least one of the following : glucose, sucrose, glycerol, succinate, citrate, te, malate, lactate, or ethanol; and - providing lactose to the cultivation medium with lactose.

In a subsequent step, the thus produced fucosylated oligosaccharide may be retrieved or obtained from the medium the host cell is cultivated in.

The step of growing and cultivating the genetically modified host cell and the step of adding lactose to the cultivation medium can be performed such, that the genetically modified host cell is first cultivated for a certain period of time, and, in a subsequent step after this first cultivation time, lactose is provided by adding it to the medium the host cell is cultivated in; alternatively, e may be provided from the beginning of the cultivation time of the genetically modified host cell, in a certain amount, and may be constantly added in a n amount. Alternatively lactose can be generated internally.

The object is further solved by a genetically modified prokaryotic host cell and by its use for the production of a fucosylated oligosaccharide, which host cell has been genetically modified such, that at least (i) the activity of a fructosephosphate- converting , which in the unmodified host cell is at a regular level, is lowered or abolished and/or by having increased the activity of a fructose—6-phosphate generating enzyme in the host cell; (ii) at least one gene encoding an enzyme necessary for the de novo synthesis of GDP-fucose is pressed; (iii) an exogenous gene encoding a fucosyltransferase, preferably an alpha-1,2—fucosyltransferase and/or alpha-1,3— fucosyltransferase, is expressed, preferably overpexressed, in the host cell. ally, the host cell has been genetically r modified (iv) to ex- press a gene encoding a protein enabling or facilitating the transport of the desired fucosylated oligosaccharide into the medium the host cell is cultivated in; and/or (v) to express an exogenous gene encoding a bifunctional L—fucokinase/L-fucose phate guanylyltransferase; and/or (vi) to have inactivated or deleted genes encoding a se- isomerase and L-fuculose-kinase; and/or (vii) to have inactivated or disrupted genes coding for enzymes of the colanic acid synthesis; and/or (viii) to express a lactose per— mease; and/or (ix) to have inactivated or deleted endogenous beta-galactosidase genes; and/or (x) to express an exogenous regulable beta-galactosidase gene; and/or (xi) to overexpress ous genes for metabolizing galactose, and/or (xii) to express an exogenous gene encoding an enzyme exhibiting fructose—1,6-bisphosphate phosphatase activity.

Also, herein ed is a method for the production of fucosylated oli- gosaccharides using a genetically modified prokaryotic host cell, the method comprising the steps of: - providing a prokaryotic host cell, which has been genetically modified, such, that at least (i) the fructosephosphate pool in said genetically modified host cell is increased by having lowered or abolished the activity of a fructose phosphate—converting enzyme, which in the unmodified host cell has a regular lev- el, or by having increased the activity of a fructosephosphate ting en— zyme; that (ii) at least one gene encoding an enzyme necessary for the de novo synthesis of GDP-fucose is overexpressed in the host cell; (iii) an exogenous gene encoding an alpha-1,2-fucosyltransferase and/or alpha-1,3-fucosyltransferase is expressed in the host cell; - cultivating said genetically ed host cell in a ation medium containing a carbon and energy source that is selected from at least one of the following : glucose, sucrose, glycerol, succinate, e, pyruvate, malate, lactate, or ethanol; and - ing lactose to the cultivation medium with e.

The objects underlying the invention are completely solved in this way.

With the method according to the invention, as well as with the genet- ically modified host cell employed in the method, it is possible to e fucosylated accharides at a titer ing 50 g/L, and even 100 g/L, and even more 150 g/L, thus providing a successful tool for the large-scale and, thus, industrial-scale fermentative production of fucosylated oligosaccharides.

Presently, and as generally understood in the state of the art, a "fucosyl- ated oligosaccharide" is a fucosylated oligosaccharide as found in human milk, i.e. an oligosaccharide that is carrying a fucose—residue. Preferably, the fucosylated oligosaccha— ride is one that is selected from 2’-fucosyllactose, 3-fucosyllactose or difucosyllactose.

Also, a "genetically ed prokaryotic host cell" presently means a prokaryotic cell whose genetic material has been altered using genetic engineering WO 77892 techniques. E.g., the host cell has been genetically modified, such, that either endoge- nous nucleic sequences naturally occurring in said host cell have been deleted, interrupt- ed or otherwise influenced so that their expression is modified, i.e. abolished, lowered, suppressed, enhanced, or similar, and/or exogenous nucleic acids, i.e. nucleic acids that are foreign to said host cell, have been introduced into the host cell to be sed, e.g. under control of an controllable promoter, in the host cell. In this connection, such genet- ically modified host cells are also called "recombinant host cells". For example, a subject prokaryotic host cell is a genetically modified prokaryotic host cell, by virtue of introduction into a suitable prokaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell.

Accordingly, the term "recombinant", as used herein with reference to a bacterial host cell tes that the bacterial cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous c acid (Le, a sequence "foreign to said cell"). Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by cial means. The term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified t removing the nucleic acid from the cell; such modifications include those obtained by gene ement, pecific muta- tion, and related techniques. Accordingly, a "recombinant polypeptide" is one which has been produced by a recombinant cell. A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is one that originates from a source foreign to the particular host cell (e.g. from a different species), or, if from the same source, is modified from its al form. Thus, a heterologous c acid operably linked to a promoter is from a source different from that from which the er was derived, or, if from the same , is modified from its original form. The heterologous sequence may be stably introduced, e.g. by transfection, transformation, conjugation or transduction, into the genome of the host microorganism cell, n techniques may be applied which will depend on the host cell and the sequence that is to be introduced. Various techniques are known to a person skilled in the art and are, e.g., sed in Sambrook et al., Molecular Cloning: A tory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989).

Accordingly, a "genetically modified yotic host cell" is presently understood as a prokaryotic cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.

The nucleic acid sequences as used in the t invention, may, e.g., be comprised in a vector which is to be stably transformed/transfected or othen/vise introduced into host microorganism cells.

A great variety of expression systems can be used to express the genes in the invention. Such vectors include, among others, chromosomal, episomal and virus— d vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast es, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system ucts may contain control regions that regulate as well as er expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and to size a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine ques, such as, for example, those set forth in Sambrook et al., supra.

The art is rich in patent and literature publications relating to "recombi- nant DNA" ologies for the isolation, synthesis, purification and amplification of genetic materials for use in the transformation of selected host organisms. Thus, it is common knowledge to transform host sms with "hybrid" viral or circular plasmid DNA which includes selected exogenous (i.e. foreign or "heterologous") DNA ces.

A person skilled in the art will know a y of methods to achieve "hybrid" vectors for use in the transformation of a selected host organism.

The term "nucleic acid sequence encoding..." generally refers to any polyribonucleotide or oxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, and generally represents a gene which encodes a certain p- tide or protein. The term includes, without limitation, single- and double-stranded DNA, DNA that is a e of - and double-stranded regions or single-, double- and triple- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single—stranded or, more typically, double-stranded, or -stranded regions, or a mixture of single- and double-stranded regions. The term also encompasses polynucleo— tides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an ion sequence or g) together with additional regions that also may contain coding and/or non—coding sequences.

As used herein, the term vating" means growing and/or incubating a bacterial cell in a medium and under conditions permissive and suitable for the tion of the desired oligosaccharide(s). A couple of suitable bacterial host cells as well as mediums and conditions for their cultivation will be readily available for one skilled in the art upon reading the disclosure of this invention in connection with the skilled person's technical and expert background. it is to be understood that with the invention as disclosed herein, the production of one or more oligosaccharides as defined herein is possible, as long as the respective c acids encoding the relevant proteins/enzymes as disclosed herein are sed in the ce||(s).

As used herein, the term "recovering" means isolating, harvesting, puri- fying, collecting or othenNise separating from the host microorganism culture the oligosac— charide produced by the host microorganism according to the invention.

According to an embodiment of the method and the use of the invention, the fucosylated ologosaccharide to be produced is ed from at least one of 2’- fucosyllactose, 3—fucosyllactose or difucosyllactose.

According to an ment of the method of the invention, the prokar- yotic host cell is selected from the group consisting of bacterial host cells, preferably selected from an Escherichia coli strain, a Lactobacil/us s, or a Corynebacterium glutamicum strain.

Preferably, the host cell is a ial host cell selected from an Esche- richia coli, bacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Lactoba— cil/us casei, Lactobacillus acidophilus, Lactobacil/us he/veticus, Lactobacillus delbrueckii, Lactococcus lactis cell. A person skilled in the art will be aware of further bacterial strains when g the present disclosure.

Presently, and as generally understood, the expression "fucosyltransfer- ase" is understood as an enzyme that transfers an L-fucose sugar from a GDP-fucose (guanosine diphosphate—fucose) donor substrate to an acceptor substrate to form the fucosylated oligosaccharide. The acceptor substrate, in the present invention. is an oligosaccharide. Also, fucosyltransferases not only catalyze fucosylation in the presence of glycan acceptors, but can also hydrolyze GDP-L—fucose when no acceptor substrate is available.

Accordingly, the terms "alpha—1,2-fucosyltransferase" or "fucosyltrans- ferase" or a nucleic acid/polynucleotide encoding an -1,2—fucosyltranferase" or "fucosyltransferase" refer to a yltransferase that catalyzes the transfer of the fucose moiety from a donor ate, for example, GDP-fucose, to an acceptor molecule in an alpha-1,2—linkage. The terms "alpha-1,3-fucosyltranferase" or "fucosyltransferase" or a nucleic acid/polynucleotide encoding an "alpha-1,3—fucosyltranferase" or yltransfer- ase" refer to a glycosyltransferase that catalyzes the transfer of the fucose moiety from a donor substrate, for example, cose, to an acceptor molecule in an alpha-1,3- linkage. The acceptor molecule can be, e.g., lactose, 2’-fucosyl|actose, 3—fucosyllactose, 3’-sialyllactose, 6’-sialy|lactose, lacto-N-tetraose, lacto-N-neotetraose or a derivative thereof.

According to the invention, the ous gene encoding a fucosyl- transferase is selected from a gene expressing a protein exhibiting an alpha-1,2- ltransferase activity, a gene expressing a protein exhibiting an alpha-1,3- fucosyltransferase ty, or a gene expressing an alpha-1,2—fucosyltransferase as well as an alpha—1,3-fucosyltransferase activity.

According to preferred embodiments, for the synthesis of 2’- fucosyllactose a le alpha—1,2-fucosyltransferase is expressed, for the synthesis of 3- fucosyllactose a suitable alpha-1,3—fucosyltransferase is expressed, for the synthesis of 2’,3-difucosyllactose, both, a suitable alpha—1,2-fucosyltransferase and an alpha-1,3- ltransferase or at least one gene encoding for a n exhibiting an alpha-1,2— as well as an alpha—1,3-fucosyltransferase ty is expressed.

Non-limiting examples for fucosyltransferases which can be used ac- cording to the invention and which shall be part of the invention are, e.g., bacterial fucosyl- transferases, and preferably an alpha-1,2-fucosyltransferase, and more preferably the alpha-1,2-fucosyltransferase encoded by the wbgL gene of E. co/i:O126, or the alpha-1,2- fucosyltransferase encoded by the fucT gene of Helicobacter pylori, or an 1,3- fucosyltransferase, and more preferably an 1,3-fucosyltransferase from Akkerman- sia muciniphila, Bacteroides fragilis, H. pylori, or H. hepaticus. Preferably, a yl- transferase is used or variants thereof which is disclosed in EP 2 479 263 A1 or from EP 2 439 264 or in made subject matter of this invention.

A "Variant" as the term is used , is a polynucleotide or polypeptide that s from a reference cleotide or polypeptide, in particular an enzyme as mentioned and used herein, respectively, but retains the essential (enzymatic) properties of the reference polynucleotide or polypeptide. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid tutions, additions, ons, s and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference ptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encod- ed by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic t, or it may be a variant that is not known to occur naturally. Non—naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct sis, and by other recombinant methods known to the persons skilled in the art.

Within the scope of the present invention, also nucleic ac- id/polynucleotide and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs are comprised by those terms, that have an amino acid sequence/nucleic acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or r amino acid ce identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a ptide as mentioned herein, e.g. to an fructosephosphate-converting enzyme, in particular phosphofructokinase A, glu— cosephosphate isomerase, fructosephosphate aldolase, a transketolase, e.g. tktA, tktB, or a transaldolase, e.g. ta/A, talB, a fructose—1,6—bisphosphate phosphatase, as used herein, to the phosphomannomutase, preferably manB, a mannosephosphate guano- syltransferase, preferably manC, a nnose—4,6-dehydratase, preferably gmd, and a fucose synthase, ably wcaG, to a fucosyltransferase as used herein, to a fructose-1,6-bisphosphate phosphatase, preferably the functional active variant of the fructose—1,6-bisphosphate phosphatase (fbpase) from Pisum sativum, to the sugar efflux transporter, e.g. 001_9420 or SetA, and to the lactose permease, e.g. LacY, used herein.

Accordingly, a "functional fragment" of any of the genes/proteins dis- closed therein, is meant to designate sequence variants of the genes/proteins still retain- ing the same or somewhat lesser activity of the gene or protein the respective nt is derived from.

As defined herein, the term "endogenous" herein and generally within the field means that the nucleic acid encoding for an enzyme of st is originating from the bacterial host cell and has not been introduced into said host cell, whereas an "exoge- nous" or "recombinant" nucleic acid has been introduced into said host cell and does not originate from said host cell.

According to another embodiment, the nucleic acid/gene is gous or heterologous. tly, and as generally understood in the relevant field, the expres- sion "homologous" refers to a nucleic acid sequence/gene that encodes for a specific product or products and is derived from the same species, in which said nucleic acid sequence is inserted. ingly, the term "heterologous" refers to a nucleic acid se— quence/gene encoding for a specific product or ts and being derived from a species other than those in which said nucleic acid sequence/gene is inserted.

According to another embodiment, the host cell of the invention further comprises control sequences enabling the controlled overexpression of endogenous or exogenous/recombinant nucleic acid sequences/genes. As defined above, the term ol sequence" which herein is synonymously used with the expression "nucleic acid/gene expression control sequence", comprises promoter sequences, signal se- quence, or array of transcription factor binding sites, which sequences affect transcription and/or translation of a c acid sequence or gene operably linked to the control sequences.

Presently, the term "operably linked" as used herein, shall mean a func- tional linkage between a nucleic acid/gene expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second c acid sequence or gene, wherein the sion control ce affects transcrip- tion and/or translation of the nucleic acid corresponding to the second sequence. Accord- ingly, the term "promoter" designates DNA sequences which usually "precede" a gene in a DNA polymer and provide a site for initiation of the transcription into mRNA. "Regulator" DNA sequences, also usually "upstream" of (Le, preceding) a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcriptional tion.

Collectively referred to as ter/regulator" or ol" DNA sequence, these se— s which precede a selected gene (or series of genes) in a functional DNA polymer cooperate to determine whether the transcription (and eventual sion) of a gene will occur. DNA sequences which "follow" a gene in a DNA polymer and provide a signal for termination of the transcription into mRNA are referred to as transcription "terminator" sequences.

As outlined already further above, the nucleic acid sequence/gene which is used according to the invention, may, e.g., be comprised in a vector which is to be stably transformed/transfected into bacterial host cells. The definitions and detailed description for recombinant production as outlined above shall apply for this paragraph.

In some embodiments, the c acid sequence/gene is placed under the control of an inducible er, which is a er that directs expression of a gene where the level of expression is alterable by environmental or pmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and als. Such promoters are referred to herein as ible" promoters, which allow one to control the timing of expression of the proteins used in the present ion. For E. coli -and other bacterial host cells, inducible promoters are known to those of skill in the art.

Throughout the invention, the expression "gene" is meant to represent a linear sequence of nucleotides (or a nucleic acid sequence; see above) along a segment of DNA that provides the coded instructions for synthesis of RNA, which, when translated into protein, leads to the expression of a protein/peptide. The protein/peptide may — as in the present invention — have certain enzymatic functions. A "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a ular nucleic acid ce includes the complementary sequence thereof.

The term ic acid sequence encoding..." or "gene(s) encod- ing/coding for..." generally refers to any polyribonucleotide or oxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, and generally repre- sents a gene which encodes a certain polypeptide or protein. The term includes, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions or single—, double- and triple-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single— and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more lly, double-stranded, or triple-stranded regions, or a e of single- and double-stranded regions. The term also asses polynucleotides that e a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) er with additional regions that also may contain coding and/or non-coding sequences.

Accordingly, in the present invention, the terms "gene" and "nucleic acid sequence" are used interchangeably.

Further, as used herein term ity" when referring to an enzyme is meant to comprise any molecule displaying enzymatic activity, in particular a protein, and acting as a catalyst to bring about a specific mical reaction while remaining un- changed by the reaction. In ular, proteins with enzymatic activities are meant to be comprised by this term, which are able to convert a substrate into a product.

In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called ts. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only a few reac- tions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Accordingly, when, ing to the invention, the activity of an enzyme is "abolished" or "lowered" the enzyme does not have the activity it has if the enzyme or its sion is unmodified, i.e. the activity, in that case, is abolished or lowered compared to the unmodified /enzyme sion.

The inventors of the present invention were able to provide for a method and a genetically modified host cell, by means of which fucosylated oligosaccharides could be produced with a product titer exceeding 100 g/L.

According to one embodiment of the invention of the method or the host cell of the invention, the activity of a fructose—6-phosphate—converting enzyme, which in the unmodified host cell is - where able —at a regular level, is lowered or abolished.

When using E, coli as a host cell, it is preferred in the method and the host cell according to the invention the fructosephosphate converting enzyme is selected from the group consisting of phosphofructokinase, preferably phosphofructokinase A (PfKA), glucose—6- phosphate isomerase, fructosephosphate aldolase, a transketolase, preferably tktA, MB, or a ldolase, preferably ta/A, talB, which fructose—6-phosphate converting enzyme, being vise present and active in the unmodified E. coli host cell, has been modified such that its activity is lowered or abolished.

Pka is efficiently phosphorylating fructosephophate to fructose-1,6- bisphosphate. Fructose-6—phosphate is the branching point in glycolytic, and gluconeo- genic ys, and in the synthesis of GDP—L—fucose that starts from the ManA cata- lyzed ization of fructosephosphate to mannosephosphate. When growing E. coli on a gluconeogenic substrate like glycerol, the phosphorylation of fructose phosphate by Pka is a highly ATP consuming treadmill reaction and, in addition, it es with ManA for the substrate.

According to an embodiment of the method and the host cell of the in- vention, the se-6—phosphate generating enzyme is fructose-1,6-bisphosphate phosphatase, the activity of which can be increased to increase the pool of fructose-6— phosphate.

According to another embodiment of the invention, at least one gene en— coding an enzyme necessary for the de novo synthesis of GDP-fucose is overexpressed in the host cell.

GDP-fucose, as mentioned above, serves as L—fucose-donor for the re- action of the fucosyltransferase transferring L-fucose to an acceptor substrate to form the fucosylated oligosaccharide.

With the prokaryotic host cell genetically modified to internally e GDP-fucose, an external addition of L-fucose, which can be converted to GDP-fucose via the salvage pathway, is not needed, since the host cell effectively produces GDP—fucose needed for the fucosylation-process of the desired oligosaccharide.

The at least one gene one encoding an enzyme necessary for the de novo synthesis of cose and being overexpressed in the host cell can be an endog- enous gene or an ous gene, which can be integrated into the genome of the host cell.

In an embodiment of the present invention, the exogenous genes encod- ing the enzymes necessary for the de novo synthesis of GDP-fucose are a gene coding for a phosphomannomutase, preferably manB, a gene coding for amannosephosphate guanosyltransferase, preferably manC, a gene coding for a GDP-mannose-4,6- dehydratase, ably gmd, and a gene coding for a GDP-L-fucose se, preferably wcaG.

According to an embodiment of the invention, and as mentioned further above, at least one exogenous gene encoding a ltransferase is selected from a gene expressing a protein that exhibits an alpha-1,2—fucosyltransferase activity and/or an alpha-1,3-fucosyltransferase activity. In this connection it is particularly preferred if the 1,2-fucosyltransferase is selected from the group consisting of wbgL of E. co/i20126 or the alpha—1,2-fucosyltransferase encoded by the fucT2 gene of Helicobacter pylori, and if the gene encoding an alpha-1,3—fucosyltransferase is selected from the group consisting of alpha-1,3-fucosyltransferases of the species Akkermansia muciniphi/a and Bacteroides fragilis, Helicobacter pylori or Helicobacter hepaticus.

According to another embodiment of the invention it is preferred if the host cell is further genetically ed: (i) to express an gene, preferably an exogenous gene, encoding a protein enabling or tating the export of the desired fucosylated oligosaccharide into the culture medium; and/or (ii) to express an ous gene encoding a bifunctional L-fucokinase/L—fucose 1-phosphate guanylyltransferase; and/or (iii) to exhibit mutated or d genes fucl and fucK leading to lowered or abolished activities of the L-fucose-isomerase (Fucl) and L-fuculose-kinase (FucK), (iv) and/or to have inactivated or ted genes ng enzymes of the colonic acid synthesis; and/or (v) to express, ably overexpress, an endogenous and/or exogenous per— mease for the import of lactose; and/or (vi) to have inactivated or deleted endogenous beta-galactosidase genes; and/or (vii) to express a gene encoding beta-galactosidase, preferably an exogenous regulable beta-galactosidase; and/or (viii) to overexpress an endogenous and/or exogenous gene coding for a se—1,6-bisphosphate phosphatase.

With the additional genetic modification as indicated above, the method for producing a fucosylated oligosaccharide could be even more improved.

With the deletion or inactivation of the genes encoding se — ase (e.g., Fuel) and L—fuculose-kinase (e.g., FucK), the lism of intracellular fucose can be avoided.

With the disruption, deletion or inactivation of genes encoding enzymes of the colonic acid biosynthesis (e.g., in E. coli as host cell, the wcaJ gene that catalyzes the first step of colanic acid synthesis), the intracellularly production of colanic acid, which otherwise might compete with the fucosyltransferase on for the substrate GDP-L- fucose, is prevented.

According to a preferred embodiment, the protein that enables or facili- tates the export of the desired fucosylated oligosaccharide into the culture medium is a the sugar efflux transporter preferably selected from yber00001_9420 and E. coli SetA.

According to a preferred embodiment, the gene encoding a bifunctional L-fucokinase/L-fucose phate guanylyltransferase is fkp from Bacteroides fragilis.

According to a preferred embodiment, the fructose-1,6-bisphosphate phosphatase is encoded by a gene which is a functional active t of the fructose—1,6- bisphosphate phosphatase (fbpase) from Pisum sativum.

According to a preferred embodiment, the lactose permease is E. coli LacY.

With the expression of a gene encoding a tional L-fucokinase/L- fucose 1-phosphate guanylyltransferase that catalyzes the synthesis of GDP—L-fucose, eg. of the Bacteroides fragilis fkp gene, the formation of free L-fucose that could accumu- late as a uct following the hydrolysis of GDP-L-fucose is prevented, thereby rescuing free L-fucose for the synthesis of the desired fucosylated oligosaccharide.

According to a preferred ment, the exogenous genes for metabolizing ose are the genes comprising the ga/ETKM operon and/or gaIP from E. coli.

According to an embodiment of the invention, the genes the host cell is modified in or with are endogenous or exogenous genes.

Throughout the invention, and ng for each gene/nucleic acid that has been exogenously introduced into the host cell, it is — according to an embodiment of the present method and host cell - preferred, if at least one of the exogenous genes, preferably integrated in the host cell genome, is overexpressed, preferably upon endoge- nous or exogenous induction, or in a constitutive manner.

Accordingly, it is preferred if at least one of the following gene(s) is/are overexpressed: (i) exogenous genes encoding enzymes necessary for the de novo synthesis of GDP-fucose; (ii) an exogenous gene ng a fucosyltransferase; (iii) an exogenous gene encoding a sugar efflux transporter; (iv) an exogenous gene ng a bifunctional L-fucokinase/L-fucose 1-phosphate guanylyltransferase; (v) a lactose per- mease; (vi) an exogenous regulable gene encoding a beta-galactosidase; and/or (vii) exogenous genes for metabolizing galactose; (viii) an exogeneous gene encoding a se-1,6-bisphospahate phosphatase; the overexpression can be effected, e.g., by means of a regulable promoter that initiates ription of the gene(s), either at a certain time point or period during the cultivation or for the whole cultivation time.

Also, according to a red embodiment, the exogenous genes to be introduced in the host cell employed in the method according to the invention are integrat- ed into the genome of the host cell.

Also, according to one aspect of the ion, and unless ise de- fined, the genes the host cell is modified in/with according to the invention can also be endogenous genes, and their expression can be enhanced or increased or overex— d, or otherwise abolished or decreased.

With the inactivation or deletion of endogenous beta-galactosidase gene(s) the degradation of externally added lactose is prevented; however, since it is desirable to have lactose ed that is not metabolized and that would othenNise impede the purification of the desired fucosylated oligosaccharide, it is also preferred if an exogenous regulable gene encoding a beta-galactosidase or a mutated form of the beta- galactosidase is expressed in the host cell. E.g., the IacZQ fragment of the lacZ gene can be expressed, the expression of which, e.g., can be regulated by means of a repressor, e.g. by a ature sensitive transcriptional repressor, e.g. cl857. In this case, synthe— sis of the beta-galactosidase Q-fragment can be initiated by raising the temperature to 42°C.

A repressor, as presently and generally in the state of the art under- stood, is a DNA— or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding sor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA.

As a promoter for the exogenous beta-galactosidase alpha fragment, e.g., the E. coli BL21 (DE3) PgbA promoter can be used. The beta-galactosidase a- and Q-fragments are combined to result in an active beta-galactosidase in the cell.

In a preferred ment, the lactose is provided by adding lactose from the beginning of the cultivation in a concentration of at least 5 mM, more preferably in a concentration of more than 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM, or even more in a concentration of 300 mM or higher than 300, or 400 mM.

According to yet another embodiment, lactose is provided by adding lac- tose the cultivation medium in a concentration, such, that throughout the production phase of the cultivation a lactose concentration of at least 5 mM, more ably of at least 10, or 30 mM, is obtained.

Alternatively, e can be produced by the host cell intracellularly, as described in patent 316 A1, the content of which is herewith sively referred to and incorporated by reference.

In the method according to the invention, it is preferred if the host cells are cultivated for at least about 60, 80, 100 or about 120 hours or in a uous manner.

Thus, according to one aspect of the invention, i.e. in a uous method, a carbon source is constantly added to the medium during the cultivating step of the host cell. By constantly adding the carbon source during the cultivation step, a con- stant and effective production of the oligosaccharide is accomplished.

According to another aspect, the method according to the invention is or includes a fed—batch fermentation method, with either a constant volume tch e, where the substrate is fed without diluting the culture, or variable volume fed—batch culture, where the fermentation volume changes with the fermentation time due to the substrate feed.

As mentioned above, the t invention also concerns a genetically modified prokaryotic host cell, which host cell has been genetically ed such, that (i) the activity of a fructosephosphate-converting enzyme, which in the unmodified host cell is at a regular level, is lowered or abolished; (ii) at least one gene encoding an en- zyme necessary for the de novo synthesis of GDP-fucose is overexpressed; (iii) an exogenous gene ng a fucosyltransferase, preferably a gene encoding an alpha-1,2— fucosyltransferase and/or an 1,3-fucosyltranferase, is expressed in the cell.

As mentioned for the method above, the host cell is preferbaly selected from a Escherichia coli strain, a Lactobaci/lus strain or a Corynebacterium strain.

According to an embodiment of the host cell according to the invention, the ellular pool of fructose-G-phosphate is increased by (i) lowering or abolishing the activity of a fructose—6-phosphate ting enzyme that is selected from the group of phosphofructokinase, glucosephosphate isomerase, fructosephosphate aldolase, a transketolase, e.g. tktA, tktB, or a transaldolase, e.g. talA, taIB, or (ii) increasing the se-1,6-bisphosphate phosphatase activity.

In a preferred embodiment, the genes encoding enzymes necessary for the de novo synthesis of GDP—fucose are pressed.

In yet another preferred embodiment, the exogenous genes encoding at least one fucosyltransferase are genes encoding alpha-1,2-fucosylltransferases and/or 1,3-fucosyltransferases and are selected from wbgL from E. coli 0126 or fucT2 from Helicobacter pylori, referring to alpha—1,2—fucosylltransferases, and genes of the s Akkermansia muciniphi/a, Bacteroides fragilis, Helicobacter pylori, or Helicobacter hepaticus, referring to alpha-1,3—fucosyltransferases.

According to an embodiment, the host cell as described above is op- tionally cally r modified (iv) to express an exogenous gene encoding a sugar efflux transporter; and/or (v) to express an exogenous gene encoding a bifunctional L- fucokinase/L-fucose 1-phosphate guanylyltransferase; and/or (vi) to have inactivated or deleted genes encoding a L-fucose—isomerase and L—fuculose-kinase; and/or (vii) to have inactivated or disrupted genes encoding the UDP-glucose:undecaprenyl phosphate glucosephosphate transferase; and/or (viii) to express an exogenous lactose per— mease; and/or (ix) to have inactivated or deleted endogenous beta-galactosidase genes; and/or (x) to express an exogenous ble gene encoding a beta-galactosidase; and/or (xi) to express exogenous genes for metabolizing galactose; and/or to express an exoge- nous gene encoding a se-1,6-bisphosphare phosphatase.

The present invention also concerns the use of the genetically modified prokaryotic host cell according to the invention for the production of a fucosylated oligo- sacchafide r advantages follow from the description of the embodiments and the attached drawings.

It goes without saying that the abovementioned es and the fea- tures which are still to be explained below can be used not only in the respectively speci- fied ations, but also in other combinations or on their own, without departing from the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES Several embodiments of the invention are illustrated in the figures and explained in more detail in the following description. In the s: Fig. 1 A schematic, exemplary illustration of a genetically modified host cell to be used in the method according to the invention; Fig. 2 HPLC analyses of supernatants from glycerol grown cultures of 2’—fucosyllactose producing E. coli s by HPLC; and Fig. 3 SEQ-ID Nos. 1 to 7 DETAILED DESCRIPTION OF THE FIGURES AND EMBODIMENTS Fig. 1 shows an exemplary illustrative host cell ing to the inven- tion to be ed in the method according to the invention, with exemplary pathways for the tation of the exemplary fucosylated oligosaccharides 2’—fucosyl|actose being depicted. In figure 1, an exemplary bacterial host cell is shown that has been genetically modified according to the invention, with respect to the production of 2‘-fucosyllactose.

As can be seen from Fig. 1, glycerol is exemplary used as carbon source, while lactose is externally added. e is transported into the host cell via a permease (e.g. LacY). Glycerol is taken up into the host cell via tated diffusion through GIpF. Within the prokaryotic host cell, glycerol is converted into glyceraldehyde phosphate, which is converted to sephosphate, (i) favored by the overexpression of an exogenous gene encoding a Fbpase and (ii) with the reverse on being inhibited by the inactivation of the phosphofructokinase A (Pka). Via overexpression of exogenous enzymes necessary for the de novo synthesis of GDP-fucose, i.e. phosphomannomutase ManB, mannosephosphate guanosyltransferase ManC, GDP-mannose-4,6— dehydratase Gmd, and GDP-L-fucose synthase WcaG, GDP—L-fucose is produced.

In a next step, GDP-L—fucose, by the action of an 1,2- fucosyltransferase, e.g. ngL, reacts with the internalized lactose to produce 2— fucosyllactose, which is ed via an efflux transporter, e.g. TPYb, into the medium the host cell is cultivated in.

In Fig. 2 the s of HPLC analyses of supernatants from glycerol grown cultures of 2’-fucosyllactose ing E. coli strains by HPLC are shown.

Depicted in Fig. 2 is the HPLC e of the fermentation broth from a 2’- fucosyllactose producing strain harbouring the gene encoding the heterologous trans- porter yber00001_9420 (black) and the HPLC profile of fermentation broth of the same strain after deletion of the gene encoding the heterologous transporter yber00001_9420 (gray). Fermentation of both strains was conducted for 111 h at 28°C, using glycerol as source of carbon and energy.

Example 1 Engineering of an E. coli BL21(E)E3) strain for the production of 2’— fusosytlaotose Using E. coli BL21(DE3) as parental host a strain for the production of 2’-fucosyllactose in a whole cell thetic approach was constructed. Genomic engi— neering of the strain included gene disruption and deletion events and integration of heterologous genes.

Since 2’-fucosyllactose is synthesized from lactose, that is applied to the bacterial culture, and from GDP—L-fucose that is produced from the living cells, first the wild-type copy of the lacZ gene encoding the endogenous ctosidase was inactivat- ed by mutagenesis using mis-match oligonucleotiedes (see Ellis et aI., "High efficiency mutagenesis, repair, and engineering of somal DNA using single—stranded oligo- nucleotides", Proc. Natl. Acad. Sci. USA 98: 6742-6746 (2001). Using the same method, the gene for the arabinose-isomerase araA was disrupted.

A lacZO gene fragment was introduced under the control of the tempera- ture sensitive transcriptional repressor cl857. The lach fragment gene is expressed under the control of the E. coli BL21 (DE3) PgbA promoter in the strain, ing a LacZ+ strain.

Genomic deletions were performed by 7L Red mediated recombination according to the method of Datsenko and Warner (see "One-step inactivation of chromo- somal genes in Escherichia coli K-12 using PCR ts", Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). The genes fucl and fucK, coding for the se isomerase and the L—fuculose kinase, tively, have been deleted to prevent ation of L—fucose.

Also genes wsz—wcaJ were d. WcaJ probably encodes a UDP- glucosezundecaprenyl phosphate e—1-phosphate transferase catalysing the first step in colanic acid synthesis (see Stevenson et aI., "Organization of the Escherichia coli K—12 gene cluster sible for production of the extracellular polysaccharide colonic acid", J. Bacteriol. 178:4885—4893; (1996)); production of colanic acid would compete for GDP—fucose with the fucosyltransferase reaction.

Genomic integration of heterologous genes was performed by transposi- tion. Large gene clusters were integrated into the genome mediated by the hyperactive CQ-mutant of the mariner transposase Himar1 (see Lampe et a/., "Hyperactive transposa— se mutants of the Himar1 mariner transposon", Proc. Natl. Acad. Sci. USA 96:11428— 11433 (1999)), that was inserted into the plasmid pEcomar under transcriptional control of the Para promotor. To enhance de novo synthesis of GDP-fucose, genes encoding phos- phomannomutase (manB), mannose—1-phosphate guanosyltransferase (manC), GDP— mannose-4,6—dehydratase (gmd), and GDP-L-fucose synthase (wcaG) from E. coli K12 DH5o were overexpressed in the E. coli BL21(DE3) strain; the operon manCB was set under control of the constitutive promoter Ptet, the operon gmd, wcaG is transcribed from the constitutive PT5 promoter. The oson cassette (SEQ ID No. 1), including the gene for the dihydrofolate reductase for trime— thoprim resistance, flanked by the inverted terminal repeats ically recognized by the mariner-like t Himar1 transposase was inserted into the E. coli genome from pEcomar CQ-manCB—gmd, wcaG-dhfr.

For chromosomal integration of single genes, the EZ—Tn5TM transposase (Epicentre, USA) was used. To produce EZ—Tn5 transposomes the gene of interest together with a FRT-site flanked antibiotic resistance cassette was ied with primers that carried on both sites the 19-bp Mosaic End recognition sites (5’- CTI'ATACACATCT (SEQ ID No. 8)) for the EZ-Tn5 transposase. Using the EZ— Tn5TM transposase, the gene for the lactose importer LacY from E. coli K12 TG1 (acc. no.

ABN72583), the syltransferase gene wbgL from E. 126 (acc. no. ADN43847), and the gene yber00001_9420 encoding a sugar efflux transporter of the major facilitator superfamily from Yersinia bercovieri ATCC 43970 (acc.no. EE008298) were integrated using the respective integration cassettes: (SEQ ID No. 2), (SEQ ID No. 3), and (SEQ ID No. 4), yielding strain. The genes wbgL and yber00001_9420 were syn- thetically synthesized and codon zed (co) by GenScript Cooperation (USA). After successful integration of the IacY gene the resistance gene was eliminated from strepto— mycin resistant clones by the FLP inase encoded on plasmid pCP20 (Datsenko and Warner, "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)).

Since E. coli E3) lacks a functional gal—operon a natively regulat- ed copy of the gaIETKM operon from E. coli K was integrated into the B strain by EZ- transposition using integration cassette (SEQ ID No. 5). lntegrands were selected from MacConkey—agar containing 1% galactose as red colonies.

The resulting strain is able to metabolize the monosaccharides glucose and galactose originating from lactose hydrolysis.

Example 2 Verification of ed osyllactoae export by Yersinia bercovieri ATCC 439m sugar efflux transporter.

] Knomkflout ofybercflclm 9420 To demonstrate onality of the heterologous sugar transporter from Yersinia bercovieri ATCC 43970 the gene yber00001__9420 was deleted from strain strain E. coli E3) IacZ, araA’, fucf, fucK, wcaJ, that contained chromosomal integra- tions of manB, manC, gmd, wcaG, lacY, wbgL; and yber00001_9420 by homologous recombination according to Datsenko and Wanner (2000; see above) using the gentamy— cine resistance cassette aacC1 from plasmid pBBR-MCSS (Kovach, Elzer et al. 1995, "Four new derivatives of the broad—host—range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes", Gene 166, 6), that was inserted into gene yber00001~9420, yielding strain Ayber00001__9420.

Cultivation conditiona for z‘nfucosyllactose graduation ] The E. coli BL21 (DE3) strain harbouring the heterologous exporter 001_9420 and the Ayber00001_9420 strain were cultivated at 28°C in 3 L ferment- ers (New Brunswick, Edison, USA) starting with 800 mL mineral salts medium ning 7 g/L NH4H2PO4, 7 g/L K2HPO4, 2 g/L KOH, 0.3 g/L citric acid, 2 g/L MgSO4>< 7H20, and 0.015 g/L CaClz >< 6H20, supplemented with 1 mL/L trace element solution (54.4 g/L ammonium ferric citrate, 9.8 g/L MnClz >< 4H20, 1.6 g/L CoClz x 6H20, 1 g/L CuClz x 2H20, 1.9 g/L H3803, 9 g/L ZnSO4 >< 7H20, 1.1 g/L NazMoO4 >< 2H20, 1.5 g/L Na28e03, 1.5 g/L NiSO4 x 6H20) containing 1.5% glycerol as carbon source and the antibiotics trime- thoprim 10 ug/ml, and kanamycin 15 ug/ml. Cultivation was started with a 2.5 % (v/v) inoculum from a pre-culture grown in the same glycerol containing medium. Lactose as acceptor in the fucosyltransferase reaction was added within seven hours to obtain a concentration of 30 mM in the culture, starting at m of about 10. Lactose was then adjusted manually to in an excess of the acceptor molecule; glycerol was added continuously.

Anatysis of e supernatant and detection of co_syliactose by; ] Analysis by high performance liquid chromatography (HPLC) was per- formed using a refractive index detector (RID—10A) (Shimadzu, Germany) and a ReproSil Carbohydrate, 5 pm (250mm x 4,6mm) (Dr. Maisch GmbH, Germany) connected to an HPLC system (Shimadzu, Germany). Elution was performed isocratically with acetoni— O (68/32 (v/v)) as eluent at 35°C and a flow rate of 1.4 ml/min. 20 pl of the sample were applied to the column. 2’-fucosyllactoseconcentration was ated from a stand- ard curve. Therefore, 10% (WV) 100 mM e were added to the HPLC samples as internal standard before they were filtered (0,22 pm pore size) and cleared by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex).

Detectian of 2’~fucosyllactase in supernatants of E. coil" BLEEDEQ} cul~ tures After 111 h of fermentation at 28°C in mineral salts medium with glycerol as carbon source, 73 mM (35,6 g/L) and 25 mM (12,2 g/L) 2’-fucosyllactose were detected by HPLC in the culture supernatant of strains containing, and lacking the 001_9420 transporter gene, see Fig 2: Depicted in Fig. 2 is the HPLC e of the fermentation broth from a 2’—fucosyllactose producing strain harbouring the gene encoding the heterol- ogous transporter yber00001_9420 (black) and the HPLC profile of fermentation broth of the same strain after deletion of the gene encoding the heterologous transporter yber00001_9420 (gray). Deletion of the heterologous sugar exporter yber00001_9420 in the strain decreases the ed amount of 2’-fucosyllactose in the supernatant. That gives evidence, that indeed the orter protein enhances 2’-fucosyllactose production by faster transport of the tri—saccharide e the cell, since the genetic background despite the yber00001_9420 gene is identical in both strains. Additionally, a lower cell density was ed in the cells lacking the 2’-fucosyllactose exporter, probably due to osmotic stress caused by strong sugar accumulation inside the cells. As shown in Fig 2, the amount of 2’,3—difucosyllactose detected in the Ayber00001_9420 culture is about double than in the broth of the original strain. Increased production of 2,3- difucosyllactose, where L-fucose is transferred to 2’—fucosy|lactose by a fucosyltransferase catalyzed reaction, also suggests higher ellular concentrations of the acceptor molecule 2’-fucosyllactose in the 001_9420 knock-out strain as compared to the yber00001_9420 overexpression strain.

] In Fig. 2, the lighter lines, i.e. the grey lines, display the supernatant of the Ayber00001_9420 E. coli BL21(DE3) strain Ayber00001_9420 the black lines display the supernatant of the culture of the yber00001__9420 containing E. coli BL21(DE3).

Samples were taken after 111 h of fermentation at 28°C in mineral salts medium using glycerol as carbon source.

Example 3 Production of 2’-fucosyllactose in a tative process Fermentations were conducted in 3 L-fermenters at 30°C and at pH 7.0; the pH was regulated by titration with 25% ammonia. The strain described in example 2 was cultivated in the l salts medium described in example 2 using glycerol as source of carbon and energy. The fermenter with a starting volume of 1L was inoculated with a pre-culture cultivated in the same medium. After consumption of the 2% glycerol contained within the batch, glycerol (60% v/v) was fed continuously. Lactose in a concen- tration of 0.66 M was added in three portions (in an one hour interval) of 10 mL each when an ODSOOnm of 6 was reached. Aften/vards, e was given in a continuous flow to hold a lactose concentration of at least 10 mM in the fermenter. After 86 h of cultivation a final titer of 91.3 mM (44,6 g/L) 2’-fucosyllactose was reached. By shifting the temperature to 42°C, the B-galactosidase gene is expressed and lactose and its degradation products glucose and galactose are metabolized by the 2’-fucosyllactose production strain.

Example 4 HPLC-analysis of culture supernatant Analysis by HPLC was performed using a refractive index detector (RID- 10A) (Shimadzu, Germany) and a Waters XBridge Amide Column 3.5um (250 x 4.6mm) (Eschborn, y) ted to an HPLC system (Shimadzu, Germany). Elution was performed isocratically with 30% A: 50% (v/v) ACN in ddHZO, 0.1% (v/v) NH4OH and 70% B: 80% (v/v) ACN in ddHZO, 0.1% (v/v) NH4OH (v/v) as eluent at 35°C and at a flow rate of 1.4 ml/min. 10 pl of the sample were applied to the column, and the 2’-fucosyllactose concentration was calculated from a standard curve. Therefore, 10% (v/v) of a 100 mM sucrose solution was added to the HPLC samples as an internal standard prior to filtering (0.22 pm pore size) and clearing by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). By-products like L-fucose, 3-fucosyllactose, 2‘,3- syllactose, and fucosylgalactose were also detected using the same analysis conditions.

Example 5 ement of 2’-fucosyllactose production strain by metabolic engi- neenng ] Further improvement ning the synthesis of osyllactose by the E. coli strain was achieved by on of the pka gene, encoding the phosphofructo- kinase A. When cultivating E. coli on a gluconeogenic ate like glycerol the phos- phorylation of fructosephosphate by Pka is a highly ATP consuming treadmill reaction and, in addition, it competes with ManA for the substrate. The pka gene was deleted by homologous recombination according to Datsenko and Wanner (2000, see above) using a gentamycin resistance cassette (aacC1) that was d by lox71/66 sites (see Lambert, s et al. 2007 "Cre-lox-based system for multiple gene deletions and selectable- marker removal in Lactobacillus plantarum", Appl. Environ. Microbial. 73, 1126-113). After sful deletion the pka gene the antibiotic resistance gene was removed from E. coli genome using the Cre recombinase (see Abremski, Hoess et al. 1983, "Studies on the properties of P1 site-specific recombination: evidence for topologically ed products following recombination", Cell 32, 1301-1311) that was cloned under the control of the Pare promoter in the pKD46 (see Datsenko and Wanner, 2000) chassis.

For different fucosyltransferases besides the transferase activity a GDP- L—fucose hydrolase activity was demonstrated. Also for wbgL, the alpha—1,2— fucosyltransferase used here for 2’-fucosyllactose synthesis this ytic activity was shown (see EP3050973 A1). To rescue free L—fucose for the 2’-fucosyllactose production and to eliminate the contaminating L-fucose from the culture broth, the fkp gene, encoding the bifunctional L-fucokinase/L-fucose 1—phosphat guanylyltranferase of Bacteroides fragilis, under transcriptional control of the Ptet promoter, together with the lox71/66 flanked aacC1 gene was chromosomally integrated into the strain described in example 1 by transposition using the EZ—Tn5TM transposase, (Seq ID 6).

After successful integration the gentamycin resistance gene was removed from the genome as described above.

Example 6 Optimized fermentation p_rocess for the production of 2’-fucosyllactose Using an optimized mineral salts medium that contains 3 g/L , 12 g/L K2HPO4. 5 g/L (NH4)ZSO4, 0.3 g/L citric acid, 2 g/L MgSO4>< 7H20, 0.1 g/L NaCl and 0.015 g/L CaClz >< 6H20 with 1 mL/L trace element solution (54.4 g/L ammonium ferric citrate, 9.8 g/L MnCl2 >< 4H20, 1.6 g/L CoClz x 6H20, 1 g/L CuClz >< 2H20, 1.9 g/L H3B03, 9 g/L ZnSO4 >< 7H20, 1.1 g/L NazMoO4 >< 2H20, 1.5 g/L Na28e03, 1.5 g/L NiSO4 >< 6H20) and 2% ol as carbon source batch, the E. coli strain described in example 5 was cultivated in a 3L fermenter at 33°C. The pH was hold at 7.0 by ing 25% ammonia.

The ter was inoculated to an m of 0.1 with a pre-culture grown in the same . Lactose was added when the culture obtained an ODGOOnm of 5, to obtain a concentration of 30 mM. A tration of 20—30 mM lactose was held hout the whole tation process, regulated according to HPLC-analyses. Glycerol feeding (60% v/v) started after the glycerol in the batch was consumed with flow rates of 4.5 ml/L/h for 20 hours, followed by feeding for 33 hours with 5.7 ml/L/h and 18 hours for 7.1 ml/L/h over a period of 18 hours (feeding rates are referring to the starting volume).

Overall, after 93 h a 2’-fucosyllactose titer of 106.5 g/L (217 mM) was obtained.

Example 7 ering of an enhanced 2’~fucosyiiactose production strain by met- abolic challenging To enhance the flux of the metabolized carbon source glycerol through the gluconegentic pathway from triose—phosphates to fructosephophate to feed the GDP-L-fucose biosynthesis the genes ng the fructose-1,6-bisphosphate aldolase (fbaB) and a heterologous fructose—1,6-bisphosphate phosphatase (fbpase) from Pisum sativum were overexpressed in the strain described in example 5. The fbaB gene from E. coli BL21 (DE3) was fused with the Ptet promoter. The activity of the plasic P. sativum FBPase is allosterically regulated by a disulfide—dithiol exchange due to reduction by thioredoxins. Exchange of the cysteine residue 153 to serine results in a constitutively active enzyme. The gene encoding the chloroplastic FBPase from P. sativum (acc. No.

AAD10213) was purchased codon optimized for expression in E. coli, N-terminally tagged with a hexahistidine-tag and modified to encode the C1538 t of the enzyme from Genescript. The fbpase gene is transcribed from a T7 promoter. The cassette (Seq ID 7) was used for EZ—Tn5TM transposase mediated integration in the host strain. After removal of the gentamycin resistance gene from the E. coli genome the strain was used for 2’-fucosyllactose production.

Production of "150git. osyllactose bye, fermentation process The 2’-fucosyllactose production strain genetically modified das desribed in example 7 was cultivated in the same medium at 33°C as described in example 5.

Additionally, to the 2% glycerol batch 60 mM lactose were added initially to the fermenta- tion medium. Continuous lactose g with 0.66 M lactose was stared at an ODeoo nm of about 10. Additionally, lactose mentation was carried out with a 1 M solution.

The e concentration was kept at approximately at 30 mM. After leaving the batch phase, indicated by a rise in the dissolved oxygen level, the glycerol feed (60% v/v) started with a flow rate of 6.9 ml/L/h for 37 hours (referring to the starting volume). After- wards the feed was reduced to 9.4 ml/L/h for 19 hours, and then raised again to 7.3 ml/L/h for 19 hours. 93 hours after seeding the fermenter a 2'—fucosyllactose titer of 150,2 g/L was reached.

Example 9 Production of 3afucos¥llactose fmm glycerol Using E. coli BL21 (DE3) IacZ chaJ AfuclK with chromosomal a- tion of the genes encoding the enzymes for de novo synthesis of GDP-Fucose (ManB, ManC, Gmd, WcaG) a 3-fucosyllactose production strain was constructed.

The gene encoding the 1,3-fucosylltransefrase from Bacteroides fragilis (EP 2439264 A1) together with the gene encoding the sugar efflux transporter SetA from E. coli (U82014/0120611 A1) and a gene conferring gentamycin resistance was ated into the E. coli genome. Fermentation of the strain to produce 3- fucosyllactose was ted under ions described in example 6. Glycerol feeding started after leaving the batch phase with a feeding rate of 7.4 ml/L/h (referring to the starting volume). Lactose was added to the culture to a concentration of 33 mM, when an ODsoo nm of 30 was reached. Throughout the process, e was added to hold a con- centration of at least 10 mM in the supernatant. After 88h the process was stopped at a 3— fucosyllactose concentration in the supernatant of 30 g/L.

Claims (18)

Claims

1. Method for the production of fucosylated oligosaccharides using a genetically modified prokaryotic host cell, the method comprising the steps of: - providing a prokaryotic host cell, which has been genetically modified, such, that at least (i) the activity of a fructose-6—phosphate-converting enzyme, which in the unmodified host cell has a regular level, is lowered or abolished; (ii) at least one gene encoding an enzyme necessary for the de novo synthesis of GDP-fucose is overexpressed in the host cell; (iii) an exogenous gene, encoding an alpha-1,2—fucosyltransferase and/or alpha-1,3- fucosyltransferase, is expressed in the host cell; - cultivating and/or growing said genetically modified host cell in a cultivation medi- um from a carbon and/or energy source that is selected from at least one of the following: ol, succinate, malate, te, lactate, ethanol, e; and w providing lactose to the cultivation medium; thereby producing the fucosylated accharide obtainable from the medium the host cell is cultivated in.

2. The method of claim 1, wherein the fucosylated oligosaccharide is selected from the group ting of 2’-fucosyllactose, 3-fucosyllactose or difucosyllactose.

3. The method of claim 1 or 2, wherein the host cell is selected from the group consisting of bacterial host cells, preferably selected from an Escherichia coli strain, a Lactobaci/Ius s or a Corynebacterium glutamicum strain.

4. The method of any of the preceding , wherein the fructosephosphate pool in the cell is increased by lowering or abolishing the activity of a fructosephosphate converting enzyme that is ed from the group of phosphofructokinase, glucose phosphate isomerase, fructose-G-phosphate aldolase, a transketolase, or a transaldolase, and/or by increasing the activity of a fructose-1,6-bisphosphate phosphatase.

5. The method of any of the preceding claims, wherein the genes encoding enzymes necessary for the de novo synthesis of GDP-fucose are a phosphomannomutase encod- ing gene, preferably manB, a mannose—1—phosphate guanosyltransferase encoding gene, preferably manC, a GDP-mannose-4,6-dehydratase encoding gene, preferably gmd, and a GDP—L-fucose synthase encoding gene, preferably wcaG.

6. The method of any of the preceding claims, n the gene encoding at least one fucosyltransferase exhibits an alpha—1,2-fucosyltransferase and/or alpha-1,3 fucosyl— transferase activity.

7. The method of claim 6, n the gene ng an alpha-1,2- fucosyltransferase is selected from the group consisting of wbgL from E. coli 0126 or fucT2 from Helicobacter pylori.

8. The method of claim 6, wherein the gene encoding an alpha-1,3- fucosyltransferase is selected from the group consisting of alpha—1,3-fucosyltransferase genes of the s Akkermansia muciniphi/a, Bacteroides fragi/is,He/icobacter , or Helicobacter hepaticus.

9. The method of any of the preceding , n the host cell is further genet- ically modified to express a gene encoding a protein which enables or tates the export of the desired fucosylated oligosaccharide into the culture medium.

10. The method of any of the preceding claims, n an endogenous or exogenous permease for the import of lactose is overexpressed.

11. The method of any of the preceding claims, wherein the genes the host cell is modified in or with are endogenous or exogenous genes. WO 77892

12. The method of any of the ing claims, wherein at least one of the genes the host cell is modified in or with is overexpressed upon endogenous or exogenous induction or in a constitutive manner.

13. The method of claim 9, wherein the gene encoding a protein which enables or facilitates the export of the desired fucosylated oligosaccharide is a sugar efflux trans- porter, preferably selected from 001_9420 and SetA.

14. The method of any of claims 4 to 13, wherein the fructose—1,6-bisphosphate phosphatase is encoded by a gene which is a functional active variant of the fructose-1,6- bisphosphate atase (fbpase) from Pisum sativum.

15. The method of any of claims 10 to 13, wherein the e se is E. coli LacY.

16. The method of any of the preceding claims, wherein the providing of lactose is accomplished by adding lactose from the beginning of the cultivating in a concentration of at least 5mM, preferably in a con-centration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration > 300 mM.

17. The method of any of the preceding claims wherein providing of lactose is accom— plished by adding lactose to the cultivation medium in a concentration, such, that through- out the production phase of the cultivation a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained.

18. The method of any of the preceding claims, wherein the host cells are cultivated for at least about 60, 80, 100, or about