RU2790445C2 - Improved method for production of fucosylated oligosaccharides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the production of 2'-fucosyllactose, using a genetically modified prokaryotic host cell. The host cell is genetically modified in such a way that at least (1) activity of enzyme converting fructose-6-phosphate is reduced or suppressed and/or by increase in activity of fructose-1,6-biphosphate-phosphatase; (2) at least one gene encoding enzyme necessary for synthesis of GDP-fucose de novo is super-expressed in the host cell; (3) an exogenous gene encoding alpha-1,2-fucosyltransferase is expressed in the host cell; and (4) a gene encoding fructose-1,6-biphosphate-aldolase is super-expressed. The specified host cell is cultivated in a cultural medium containing glycerin, and supply of lactose is provided. Fucosylated oligosaccharide is obtained, which can be extracted from the medium.

EFFECT: invention allows for biosynthesis of 2'-fucosyllactose in a concentration exceeding 100 g/l.

15 cl, 3 dwg, 9 ex

Description

The present invention relates to a method for producing fucosylated oligosaccharides using a genetically modified prokaryotic host cell, as well as to a host cell used in this method and its use for producing fucosylated oligosaccharides at high concentrations.

PRIOR ART

Human breast milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. The most predominant fraction is represented by carbohydrates, which in turn can be divided into lactose and more complex oligosaccharides (breast milk oligosaccharides, HMO). While lactose is used as an energy source, complex oligosaccharides are not metabolized in the infant's body. The fraction of complex oligosaccharides is up to 20% inclusive of the entire carbohydrate fraction and consists of more than 200 different oligosaccharides. The presence and concentration of these complex oligosaccharides are specific to humans and therefore they cannot be found in high amounts in the milk of other mammals.

To date, approximately 200 structurally distinct HMOs have been identified and their many useful properties have been described. HMOs are not digested in breastfed infants but provide a valuable source of carbon and energy for beneficial Bifidobacteria, Lactobacillus and Bacteroides bacteria in the gut, leading to a predominance of these bacteria in the gut and helping to suppress the growth of pathogens, thus preventing infections of the intestinal epithelium. However, in addition, HMOs directly bind to pathogenic bacteria, protozoa, and viruses, blocking pathogen-host interactions by mimicking the glycan chains of cell surface receptors, and thereby protecting the breastfed infant from infectious diseases.

The most common oligosaccharide is 2'-fucosyllactose. Other common HMOs found in breast milk are 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose. In addition to these neutral oligosaccharides, acidic HMOs can be found in breast milk, such as, for example, 3'-sialyllactose, 6'-sialyllactose and sialyllacto-N-tetraose a, b and c or sialyllacto-N-fucopentaose II and so on. These structures are closely related to epitopes of glycoconjugates on the surface of epithelial cells, with antigens of the histogroups of the blood of the Lewis system, and the structural homology of HMO with epitopes of epithelial cells explains the protective properties against bacterial pathogens.

Due to their useful properties, HMOs are approved for inclusion as ingredients in infant formulas and other food products, which makes it necessary to obtain HMOs in large quantities up to the scale of large-scale production.

Due to the limited availability of raw materials and the difficulty in obtaining purified fractions of individual breast milk oligosaccharides, chemical methods have been developed to obtain some of these complex molecules. However, the chemical and biocatalytic approaches have not been commercially viable and, in addition, certain chemical pathways for the synthesis of human milk oligosaccharides involve some harmful chemicals that pose a risk of contamination of the final product.

Due to the difficulties encountered in the chemical synthesis of human milk oligosaccharides, several methods have been developed using enzymes and fermentation approaches. To date, for some of the HMOs, such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose II, 3'-sialyllactose and 6'-sialyllactose, enzymatic approaches have been developed using mostly genetically modified bacterial strains, such as a recombinant Escherichia coli strain.

However, using even the most effective currently available methods based on bacterial fermentation, it is not possible to achieve or hardly achieve concentrations of HMO in the culture fluid of more than 20 g/l. Generally, concentrations of 50 g/L should be exceeded for industrial scale applications, although 100 g/L is a more desirable concentration.

Thus, the object of the present invention is to provide an improved fermentation process by which it is possible to carry out the biosynthesis of fucosylated oligosaccharides, in particular 2'-fucosyllactose, at a concentration exceeding 100 g/l.

BRIEF DESCRIPTION OF THE INVENTION

This and other problems are solved by developing a method for obtaining fucosylated oligosaccharides using a genetically modified host prokaryotic cell, which includes the following steps:

- providing a host cell genetically modified such that at least (1) the activity of the enzyme that converts fructose-6-phosphate, which is at a normal level in an unmodified host cell, is reduced or suppressed; (2) at least one gene encoding an enzyme necessary for the synthesis of GDP-fucose (GDF stands for guanosine diphosphate) de novo is overexpressed in the host cell; (3) an exogenous gene encoding a fucosyltransferase, preferably alpha-1,2-fucosyltransferase and/or alpha-1,3-fucosyltransferase, is expressed, preferably overexpressed, in the host cell;

- culturing said genetically modified host cell in a culture medium containing a source of carbon and energy selected from at least one of the following sources: glucose, sucrose, glycerol, succinate, citrate, pyruvate, malate, lactate or ethanol; And

- ensuring the supply of lactose to the culture medium with lactose.

In a subsequent step, the fucosylated oligosaccharide thus formed can be extracted or obtained from the medium in which the host cell is cultured.

The step of growing and culturing the genetically modified host cell and the step of adding lactose to the culture medium can be carried out in such a way that, first, the genetically modified host cell is cultured for a certain period of time, and in the subsequent step, after the period of such initial cultivation, lactose is supplied by adding it to the culture medium. the medium in which the host cell is cultured; alternatively, lactose can be added in a certain amount at the beginning of the culture period of the genetically modified host cell, and can be continuously added in a certain amount. Alternatively, lactose can be produced within the medium itself.

This object is also achieved by a genetically modified prokaryotic host cell and by using it to produce a fucosylated oligosaccharide, wherein the host cell is genetically modified such that at least (1) the activity of a fructose-6-phosphate converting enzyme, which in an unmodified cell the host is at the usual level, reduced or suppressed and/or there is an increased activity of the enzyme that catalyzes the formation of fructose-6-phosphate in the host cell; (2) at least one gene encoding an enzyme required for de novo synthesis of GDP-fucose is overexpressed; (3) an exogenous gene encoding a fucosyltransferase, preferably alpha-1,2-fucosyltransferase and/or alpha-1,3-fucosyltransferase, is expressed, preferably overexpressed, in the host cell.

It is possible that the host cell has been further genetically modified (4) to express a gene that encodes a protein that allows or facilitates the transport of the desired fucosylated oligosaccharide into the medium in which the host cell is cultured; and/or (5) for the purpose of expressing an exogenous gene encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase; and/or (6) for the purpose of inactivating or deleting genes encoding L-fucose isomerase and L-fuculose kinase; and/or (7) for the purpose of inactivating or destroying genes encoding enzymes for the synthesis of colanic acid; and/or (8) to express lactose permease; and/or (9) to inactivate or delete endogenous beta-galactosidase genes; and/or (10) for the purpose of expressing an exogenously regulated beta-galactosidase gene; and/or (11) to overexpress exogenous genes responsible for metabolizing galactose, and/or (12) to express an exogenous gene encoding an enzyme that exhibits fructose 1,6-bisphosphate phosphatase activity.

In addition, according to this invention, a method is provided for obtaining fucosylated oligosaccharides using a genetically modified prokaryotic host cell, including the steps:

- providing a prokaryotic host cell genetically modified such that at least (1) in said genetically modified host cell, the amount of fructose-6-phosphate has increased as a result of a decrease or suppression of the activity of an enzyme that converts fructose-6-phosphate, which in an unmodified cell the host is at the usual level or as a result of an increase in the activity of an enzyme that catalyzes the formation of fructose-6-phosphate; that (2) at least one gene encoding an enzyme required for de novo synthesis of GDP-fucose is overexpressed in the host cell; (3) an exogenous gene encoding alpha-1,2-fucosyltransferase and/or alpha-1,3-fucosyltransferase is expressed in the host cell;

- culturing said genetically modified host cell in a culture medium containing a source of carbon and energy selected from at least one of the following sources: glucose, sucrose, glycerol, succinate, citrate, pyruvate, malate, lactate or ethanol; And

- ensuring the supply of lactose to the culture medium with lactose.

Using this approach, the problems underlying the invention are completely solved.

Using the method according to the invention, as well as using the genetically modified host cell used in this method, it is possible to obtain fucosylated oligosaccharides at a concentration exceeding 50 g/l, and even 100 g/l, and even above 150 g/l, thus providing an efficient tool for large-scale and hence industrial enzymatic production of fucosylated oligosaccharides.

In this application and according to the general understanding of the prior art, the term "fucosylated oligosaccharide" means fucosylated oligosaccharide in the form in which it is found in breast milk, i. an oligosaccharide containing a fucose residue. Preferably, the fucosylated oligosaccharide is an oligosaccharide selected from 2'-fucosyllactose, 3-fucosyllactose, or difucosyllactose.

In addition, in the present application, the term "genetically modified prokaryotic host cell" means a prokaryotic cell whose genetic material has been changed using genetic engineering methods. For example, a host cell has been genetically modified such that either naturally occurring endogenous nucleic acid sequences present in said host cell have been deleted, ruptured, or otherwise altered in their expression, i. suppression, reduction, suppression, enhancement, or the like, and/or exogenous nucleic acids have been introduced into the host cell, i. e. nucleic acids foreign to said host cell, for expression in the host cell, eg under the control of a controlled promoter. In this regard, such genetically modified host cells are also referred to as "recombinant host cells". For example, the subject prokaryotic host cell is a genetically modified prokaryotic host cell due to the introduction into a suitable prokaryotic host cell of a heterologous nucleic acid, for example, 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 cell. owner.

Accordingly, the term "recombinant" as used herein with reference to a bacterial host cell indicates that the bacterial cell replicates a heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to specified cell). Recombinant cells may contain genes that are not found in the native (non-recombinant) form of the cell. Recombinant cells may also contain genes found in the native form of the cell, whereby the genes are modified and artificially returned to the cell. The term also encompasses cells that contain nucleic acid endogenous to that cell, modified without removing the nucleic acid from the cell; such modifications include modifications made using gene replacement, site-specific mutation, and similar techniques. Accordingly, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell. A “heterologous sequence” or “heterologous nucleic acid”, as used herein, is derived from a source that is foreign to that particular host cell (e.g., another species) or, if derived from the same source, is modified from its original form. Thus, a heterologous nucleic acid operably linked to a promoter is from a different source than the promoter, or if derived from the same source, is modified from its original form. A heterologous sequence can be stably introduced into the genome of a host microorganism cell by, for example, transfection, transformation, conjugation, or transduction, using techniques that will depend on the host cell and the sequence to be introduced. The person skilled in the art is aware of various methods, which are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Accordingly, as used herein, "genetically modified prokaryotic host cell" refers to a prokaryotic cell that has been transformed or transfected with, or susceptible to transformation or transfection with, an exogenous polynucleotide sequence.

The nucleic acid sequences used in the present invention may, for example, be incorporated into a vector that is stably transformed/transfected into host microorganism cells, or otherwise introduced into host microorganism cells.

In the present invention, a wide variety of expression systems can be used to express genes. Such vectors include, among others, chromosomal, episome, and viral vectors, e.g., vectors derived from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from their combinations, such as vectors derived from the genetic elements of plasmids and bacteriophages such as cosmids and phagemids. Expression system constructs may contain control regions that regulate as well as induce expression. Generally, any system or vector suitable for maintaining, increasing or expressing the polynucleotides and for synthesizing the polypeptide in the host cell can be used for expression in this regard. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well known and routine methods, such as those described in Sambrook et al. above.

The art is replete with patent and literature publications relating to "recombinant DNA" methodologies for isolating, synthesizing, purifying and amplifying genetic material used to transform selected host organisms. Thus, the process of transforming a host microorganism of "hybrid" viral or circular plasmid DNA comprising selected exogenous (ie, foreign or "heterologous") DNA sequences is well known. One of skill in the art is familiar with a variety of methods for producing "hybrid" vectors used to transform a selected host microorganism.

The term "nucleic acid sequence encoding..." generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, and is typically a gene encoding a particular 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 a mixture of single and double stranded regions. double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded, or more typically double-stranded or triple-stranded regions, or a mixture of single- and double-stranded regions. The term also encompasses polynucleotides comprising a single contiguous region or discontinuous regions encoding a polypeptide (e.g. interrupted by an integrated phage or an insertion sequence or edit), together with additional regions, which may also contain coding and/or non-coding sequences.

Used in this description, the term "cultivation" means the cultivation and/or incubation of a bacterial cell in the environment and under conditions that allow the production of the desired(s) oligosaccharide(s) and suitable for its(their) production. Several suitable bacterial host cells, as well as media and conditions for culturing them, will be readily available to a person skilled in the art after reading the description of the present invention, taking into account the technical and expert skill of this person.

It should be understood that when using the invention described in this application, obtaining one or more of the oligosaccharides defined in this description is possible provided that the corresponding nucleic acids encoding the relevant proteins/enzymes described in this application are contained in the cell(s) .

As used herein, the term "recovery" means isolating, collecting, purifying, accumulating, or otherwise separating the oligosaccharide produced by the host microorganism of the invention from the culture medium of the host microorganism.

According to one embodiment of the method and use of the invention, the fucosylated oligosaccharide produced is selected from at least one of 2'-fucosyl lactose, 3-fucosyl lactose, or difucosyl lactose.

According to one embodiment of the method of the invention, the prokaryotic host cell is selected from the group consisting of bacterial host cells, preferably selected from Escherichia coli strain, Lactobacillus species or Corynebacterium glutamicum strain.

Preferably, the host cell is a host bacterial cell selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactococcus lactis cells. The person skilled in the art will be aware of additional bacterial strains upon reading the description of the present invention.

In the present application and in accordance with the general understanding of the term "fucosyltransferase" is understood as an enzyme that transfers the sugar L-fucose from the donor substrate of GDP-fucose (guanosine diphosphate-fucose) to the substrate acceptor with the formation of fucosylated oligosaccharide. In the present invention, the acceptor substrate is an oligosaccharide. In addition, fucosyltransferases not only catalyze fucosylation in the presence of glycan acceptors, but can also hydrolyze GDP-L-fucose in the absence of an acceptor substrate.

Accordingly, the terms "alpha-1,2-fucosyltransferase" or "fucosyltransferase" or a nucleic acid/polynucleotide encoding(s) "alpha-1,2-fucosyltransferase" or "fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of a fucose moiety from a donor substrate, such as GDP-fucose, to an acceptor molecule to form an alpha-1,2 bond. The terms "alpha-1,3-fucosyltransferase" or "fucosyltransferase" or a nucleic acid/polynucleotide encoding(s) "alpha-1,3-fucosyltransferase" or "fucosyltransferase" refer to a glycosyltransferase that catalyzes the transfer of a fucose moiety from a substrate- donor, for example, GDP-fucose, to an acceptor molecule to form an alpha-1,3 bond. The acceptor molecule may be, for example, lactose, 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, or a derivative thereof.

According to the invention, the exogenous gene encoding fucosyltransferase is selected from a gene expressing a protein having alpha-1,2-fucosyltransferase activity, a gene expressing a protein having alpha-1,3-fucosyltransferase activity, or a gene expressing a protein having alpha-1 ,2-fucosyltransferase, as well as alpha-1,3-fucosyltransferase activity.

According to preferred embodiments, for the synthesis of 2'-fucosyl lactose, the appropriate alpha-1,2-fucosyl transferase is expressed, for the synthesis of 3-fucosyl lactose, the appropriate alpha-1,3-fucosyl transferase is expressed, for the synthesis of 2',3-difucosyl lactose, it is expressed as the appropriate alpha-1, 2-fucosyltransferase and alpha-1,3-fucosyltransferase, or at least one gene encoding a protein having alpha-1,2- and alpha-1,3-fucosyltransferase activity.

Non-limiting examples of fucosyltransferases that can be used according to the invention and which will form part of the invention are, for example, bacterial fucosyltransferases and preferably alpha-1,2-fucosyltransferase, and more preferably alpha-1,2-fucosyltransferase encoded by the wbgL gene from E coli: O126, or alpha-1,2-fucosyltransferase encoded by the fucT gene from Helicobacter pylori, or alpha-1,3-fucosyltransferase and more preferably alpha-1,3-fucosyltransferase from Akkermansia miciniphila, Bacteroides fragilis, H. pylori or H. hepaticus. Preferably, a glycosyltransferase or variants thereof are used, which are described in EP 2479263 A1, or in EP 2439264, or in WO 2010/142305, the content of which is directly related to the subject of this invention and constitutes it.

"Variant" as a term used in this specification refers to a polynucleotide or polypeptide that differs from the reference polynucleotide or polypeptide, in particular, refers to the enzyme mentioned and used in this specification, respectively, but retains the necessary (enzymatic) properties of the reference polynucleotide or a polypeptide. An exemplary polynucleotide variant differs in nucleotide sequence from another reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide sequence changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as will be discussed below. An exemplary polypeptide variant differs in amino acid sequence from another reference polypeptide. As a rule, the set of differences is limited, so that the sequences of the reference polypeptide and the variant as a whole are very similar and in many regions are identical. The amino acid sequences of the variant and the reference polypeptide may differ by one or more substitutions, one or more additions, deletions, in any combination. The substituted or inserted amino acid residue may or may not be the residue encoded by the genetic code. A polynucleotide or polypeptide variant may be of natural origin, such as an allelic variant, or may be a variant not known in nature. Non-natural variants of polynucleotides and polypeptides can be generated by mutagenesis, direct synthesis, and other recombinant techniques known to those skilled in the art.

Also included within the scope of the present invention are polymorphic variants, alleles, mutants, and cross-species homologues of nucleic acids/polynucleotides and polypeptides having an amino acid sequence/nucleic acid sequence having an amino acid sequence identity of more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher amino acid sequence identity, preferably within a stretch of at least about 25, 50, 100, 200, 500, 1000 or more amino acids, with a polypeptide mentioned herein, for example, with a fructose-6-phosphate converting enzyme, in particular, phosphofructokinase A, glucose-6- phosphate isomerase, fructose-6-phosphate-aldolase, transketolase, e.g. tktA, tktB, or transaldolase, e.g. talA, talB, fructose-1,6-bisphosphate phosphatase used in this description, with phosphomanomutase prefer flax manB, mannose-1-phosphate guanosyltransferase, preferably manC, GDP-mannose-4,6-dehydratase, preferably gmd, and GDP-L-fucose synthase, preferably wcaG, with fucosyltransferase used in this description, with fructose- 1,6-bisphosphate phosphatase, preferably a functional variant of fructose-1,6-bisphosphate-phosphatase (fbpase) from Pisum sativum, with an efflux sugar transporter, such as yberc0001_9420 or SetA, and a lactose permease, such as LacY, used in this description.

Accordingly, a "functional fragment" of any of the genes/proteins described herein is meant to mean sequence variants of the genes/proteins that still retain the same or slightly less activity of the gene or protein from which the respective fragment is derived.

As used herein, the term "endogenous", as defined herein and as is accepted in the art, means that the nucleic acid encoding the enzyme of interest originates from a host bacterial cell and is not introduced into said host cell, whereas " the exogenous" or "recombinant" nucleic acid is introduced into said host cell and is not derived from said host cell.

In another embodiment, the nucleic acid/gene is either homologous or heterologous. As used herein and as is generally understood in the relevant art, the term "homologous" refers to a nucleic acid sequence/gene that encodes a particular product or products and is derived from the same species into which said nucleic acid sequence is inserted. Accordingly, the term "heterologous" refers to a nucleic acid sequence/gene encoding a particular product or products and originating from a species other than that into which said nucleic acid sequence/gene is inserted.

According to another embodiment, the host cell of the invention further comprises control sequences allowing controlled overexpression of endogenous or exogenous/recombinant nucleic acid sequences/genes. The term "control sequence" as defined above, which is used herein as a synonym for "nucleic acid/gene expression control sequence", refers to a sequence containing promoter sequences, a signal sequence, or a set of transcription factor binding sites whose sequences affect transcription and/or or translation of a nucleic acid sequence or gene operably linked to control sequences.

As used herein, the term "operably linked" as used herein, will mean a functional relationship between a nucleic acid/gene expression control sequence (such as a promoter, signal sequence, or a set of transcription factor binding sites) and a second nucleic acid sequence or gene, wherein the control sequence expression affects the transcription and/or translation of the nucleic acid corresponding to the second sequence. Accordingly, the term "promoter" refers to DNA sequences that typically "precede" a gene" in a polymeric DNA molecule and provide a transcription initiation site in an mRNA. "Regulatory" DNA sequences, usually also located "upstream" of a gene (i.e. precursors of a gene) in a specified polymeric DNA molecule bind proteins that determine the frequency (or degree) of transcription initiation. Collectively referred to as a "promoter/regulatory" or "control" DNA sequence, these sequences "preceding" a selected gene (or series of genes) in a functional polymeric DNA molecule, act together to determine whether transcription (and possibly expression) of a gene will occur.DNA sequences that "follow" a gene in a polymeric DNA molecule and provide a transcription termination signal in mRNA are referred to as "sequences" termination of transcription.

As already indicated above, the nucleic acid sequence/gene used according to the invention can, for example, be contained in a vector that is stably transformed/transfected into host bacterial cells. The definitions and detailed description of the above preparation of recombinants apply to this paragraph.

In some embodiments, the nucleic acid sequence/gene is placed under the control of an inducible promoter, which is a promoter that directs the expression of the gene, whereby the level of expression may be altered by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemicals. Such promoters are referred to herein as "inducible" promoters, which allow you to control the timing of the expression of the proteins used in the present invention. Those skilled in the art are aware of inducible promoters for E. coli and other bacterial host cells.

Throughout the description of the invention, the term "gene" is understood to mean a linear sequence of nucleotides (or nucleic acid sequence; see above) located in a segment of DNA, providing encoded "instructions" for the synthesis of RNA, the translation of which into a protein leads to the expression of a protein/peptide. The protein/peptide may, as in the present invention, have certain enzymatic functions. "Nucleic acid" refers to a polymer composed of deoxyribonucleotides or ribonucleotides in either single or double stranded form and, unless otherwise limited, encompasses known natural nucleotide analogs that hybridize to nucleic acids in a manner similar to natural nucleotides. Unless otherwise indicated, a single nucleic acid sequence contains its complementary sequence. Unless otherwise indicated, a particular nucleic acid sequence includes its complementary sequence.

Generally, the term “nucleic acid sequence encoding…” or “gene(s) encoding/encoding…” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, and is generally a gene which codes for a specific 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 a mixture of single and double stranded regions. double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded, or more typically double-stranded or triple-stranded regions, or a mixture of single- and double-stranded regions. The term also encompasses polynucleotides comprising a single contiguous region or discontinuous regions encoding a polypeptide (e.g., interrupted by an integrated phage or an insertion sequence or edit), together with additional regions, which may also contain coding and/or non-coding sequences.

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

In addition, as used herein, the term "activity" when referring to an enzyme is intended to include any molecule that exhibits enzymatic activity and acts as a catalyst to cause a specific biochemical reaction while remaining unchanged during the reaction, in particular a protein molecule. In particular, the term is intended to cover proteins with enzymatic activity capable of participating in the conversion of a substrate into a product.

Molecules called substrates at the start of enzymatic reactions are converted into other molecules called products. Almost all chemical reactions in a biological cell require enzymes in order to proceed at rates sufficient to sustain life. Since enzymes are selective for their substrates and only speed up some of the many possible reactions, the set of enzymes produced in a cell determines the metabolic pathways that function in that cell.

Accordingly, when, according to the invention, the activity of an enzyme is "suppressed" or "lowered", the enzyme does not have the activity that it has if the enzyme or its expression is not changed, i. in this case, the activity is suppressed or reduced compared to the unchanged enzyme/enzyme expression.

The inventors of the present invention have been able to provide a method and a genetically modified host cell by which, when producing fucosylated oligosaccharides, it is possible to achieve a product concentration in excess of 100 g/l.

According to one embodiment of the invention relating to the method or host cell of the invention, the activity of the fructose-6-phosphate converting enzyme, which in an unmodified host cell, where appropriate, is normal, is reduced or suppressed. In the case of using E. coli as the host cell, it is preferred that for the method and host cell of the invention, the fructose-6-phosphate converting enzyme is selected from the group consisting of phosphofructokinase, preferably phosphofructokinase A (PfKA), glucose-6-phosphate -isomerases, fructose-6-phosphate-aldolases, transketolases, preferably tktA, tktB, or transaldolases, preferably talA, talB, wherein the fructose-6-phosphate converting enzyme is otherwise present and active in an unmodified E. coli host cell , has been modified so that its activity is reduced or suppressed.

PfkA efficiently phosphorylates fructose-6-phosphate to fructose-1,6-bisphosphate. Fructose 6-phosphate is a branch point in the glycolytic and gluconeogenic pathways and in the synthesis of GDP-L-fucose, which begins with the ManA-catalyzed isomerization of fructose 6-phosphate to mannose-6-phosphate. In the case of growing E. coli on a gluconeogenic substrate such as glycerol, fructose-6-phosphate phosphorylation by PfkA is a typical ATP-consuming reaction, and in addition there is competition with ManA for the substrate.

According to one embodiment of the method and host cell of the invention, the enzyme involved in the formation of fructose-6-phosphate is fructose-1,6-bisphosphate-phosphatase, the activity of which can be increased to increase the amount of fructose-6-phosphate.

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

GDP-fucose, as mentioned above, serves as an L-fucose donor for a fucosyl transferase-mediated L-fucose transfer reaction to an acceptor substrate to form a fucosylated oligosaccharide.

In the case of a prokaryotic host cell genetically modified to produce its own GDP-fucose, no external addition of L-fucose is required, which can be converted to GDP-fucose via the salvage pathway, since the host cell efficiently produces GDP-fucose necessary for the process of fucosylation of the desired oligosaccharide.

At least one gene encoding an enzyme required for de novo synthesis of GDP-fucose and overexpressed in the host cell may be an endogenous gene or an exogenous gene that may be integrated into the genome of the host cell.

In one embodiment of the present invention, the exogenous genes encoding the enzymes required for de novo synthesis of GDP-fucose are: a gene encoding phosphomannomutase, preferably manB, a gene encoding mannose-1-phosphate guanosyltransferase, preferably manC, a gene encoding GDP- mannose-4,6-dehydratase, preferably gmd, and the gene encoding GDP-L-fucose synthase, preferably wcaG.

According to one embodiment of the invention, and also as mentioned above, at least one exogenous gene encoding a fucosyltransferase is selected from genes whose expression results in a protein exhibiting alpha-1,2-fucosyltransferase activity and/or alpha-1,3- fucosyl transferase activity. In this regard, it is particularly preferred if the alpha-1,2-fucosyltransferase is selected from the group consisting of wbgL from E. coli: O126 or alpha-1,2-fucosyltransferase encoded by the fucT2 gene from Helicobacter pylori, and if the gene encoding alpha-1,3-fucosyltransferase is selected from the group consisting of a gene encoding alpha-1,3-fucosyltransferase from Akkermansia muciniphila and Bacteroides fragilis, Helicobacter pylori or Helicobacter hepaticus species.

According to another embodiment of the invention, it is preferred if the host cell is subjected to further genetic modification: (1) to express a gene, preferably an exogenous gene, which encodes a protein that exports or facilitates the export of the desired fucosylated oligosaccharide to the culture medium; and/or (2) for the purpose of expressing an exogenous gene encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase; and/or (3) for the purpose of expressing fucI and fucK genes having mutations or deletions, resulting in a decrease or suppression of L-fucose isomerase (FucI) and L-fuculose kinase (FucK) activities, and/or (4) with the aim of inactivating or destruction of genes encoding enzymes for the synthesis of colanic acid; and/or (5) for the purpose of expressing, preferably overexpressing, endogenous and/or exogenous lactose import permease; and/or (6) to inactivate or delete endogenous beta-galactosidase genes; and/or (7) for the purpose of expressing a gene encoding beta-galactosidase, preferably exogenously regulated beta-galactosidase; and/or (8) for the purpose of overexpressing an endogenous and/or exogenous gene encoding fructose 1,6-bisphosphate phosphatase.

With the use of additional genetic modification, as mentioned above, it is possible to further improve the process for producing fucosylated oligosaccharide.

By deleting or inactivating the genes encoding L-fucose isomerase (eg FucI) and L-fuculose kinase (eg FucK), catabolism of intracellular fucose can be avoided.

Destruction, deletion, or inactivation of genes encoding enzymes for the biosynthesis of colanoic acid (for example, the wcaJ gene that catalyzes the first step in the synthesis of colanoic acid, when using E. coli as a host cell), intracellular production of colanoic acid, which otherwise can compete in reactions involving fucosyltransferase for the substrate GDP-L-fucose.

In a preferred embodiment, the protein that exports or facilitates the export of the desired fucosylated oligosaccharide to the culture medium is an efflux sugar transporter, preferably selected from yberc0001_9420 and SetA from E. coli.

According to a preferred embodiment, the gene encoding the bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase is the fkp gene from Bacteroides fragilis.

According to a preferred embodiment, fructose 1,6-bisphosphate phosphatase is encoded by a gene that is a functionally active variant of the fructose-1,6-bisphosphate phosphatase (fbpase) gene from Pisum sativum.

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

When a gene encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyltransferase that catalyzes the synthesis of GDP-L-fucose, such as the fkp gene from Bacteroides fragilis, is expressed, the formation of free L-fucose, which can accumulate as a by-product, is prevented. product due to hydrolysis of GDP-L-fucose, thereby retaining free L-fucose for the synthesis of the desired fucosylated oligosaccharide.

According to a preferred embodiment, the exogenous genes responsible for metabolizing galactose are genes containing the galETKM and/or galP operon from E. coli.

According to one of the embodiments of the invention, the genes that are modified in the host cell or that modify the host cell are endogenous or exogenous genes.

Throughout the description of the invention and in relation to each(th) gene/nucleic acid that has been exogenously introduced into the host cell, preferably, according to one of the embodiments of the method and the host cell according to the invention, that at least one of the exogenous genes, preferably integrated into the genome host cell, was overexpressed, preferably by endogenous or exogenous induction, or in a constitutive manner.

Accordingly, it is preferred that at least one or more of the following genes is overexpressed(s): (1) exogenous genes encoding enzymes necessary for de novo synthesis of GDP-fucose; (2) an exogenous gene encoding a fucosyltransferase; (3) an exogenous gene encoding an efflux sugar transporter; (4) an exogenous gene encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase; (5) a gene encoding lactose permease; (6) an exogenously regulated gene encoding beta-galactosidase; and/or (7) exogenous genes responsible for metabolizing galactose; (8) an exogenous gene encoding fructose 1,6-bisphosphate phosphatase; however, overexpression can be caused, for example, by a regulated promoter that initiates the transcription of the given gene(s), either at a certain point in time, or during a period of time during cultivation, or during the entire time of cultivation.

In addition, according to a preferred embodiment, the exogenous genes to be introduced into the host cell used in the method of the invention are integrated into the genome of the host cell.

In addition, according to one aspect of the invention, and unless otherwise indicated, genes that are modified in the host cell/which modify the host cell can also be endogenous genes according to the invention, and their expression can be increased or increased, or can be observed overexpression, or otherwise, may be suppressed or reduced.

In the case of inactivation or deletion of the endogenous beta-galactosidase gene(s), the breakdown of externally added lactose is prevented; however, since it is desirable to have cleaved lactose that is not metabolized and which otherwise makes it difficult to purify the desired fucosylated oligosaccharide, it is also preferred if an exogenously regulated gene encoding beta-galactosidase or a mutated form of beta-galactosidase is expressed in the host cell. For example, the lacZΩ fragment of the lacZ gene can be expressed, the expression of which, for example, can be regulated by a repressor, such as a temperature-sensitive transcriptional repressor, such as cI857. In this case, the synthesis of the Ω-fragment of beta-galactosidase can be initiated by raising the temperature to 42°C.

A repressor, as used herein and as commonly understood in the relevant art, is a DNA or RNA binding protein that inhibits the expression of one or more genes by binding to an operator or appropriate silencers. The DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing the transcription of genes to form messenger RNA.

As a promoter for the alpha fragment of exogenous beta-galactosidase, for example, the PgbA promoter from E. coli BL21 (DE3) can be used. The α- and Ω-fragments of beta-galactosidase are combined to obtain active beta-galactosidase in the cell.

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

In yet another embodiment, lactose is provided by adding lactose to the culture medium at a concentration such that during the product formation phase of the culture, a lactose concentration of at least 5 mM, more preferably at least 10, 20 or 30 mM, is reached.

Alternatively, lactose can be produced intracellularly by the host cell, as described in EP 1927316 A1, the content of which is explicitly mentioned and incorporated by reference.

In the method of the invention, it is preferred if the host cells are cultured for at least about 60, 80, 100, or about 120 hours, or continuously.

Thus, according to one aspect of the invention, i. e. in the case of the continuous culture method, the carbon source is continuously added to the medium during the stage of culturing the host cell. By continuously adding a carbon source during the culture step, continuous and efficient production of the oligosaccharide is achieved.

According to another aspect, the method of the invention is or includes a fed-batch method, using either a constant volume fed-batch culture where the substrate is fed without diluting the culture, or a variable-volume fed-batch culture where the fermentation volume varies depending on the duration of the fermentation due to the feeding substrate.

As mentioned above, the present invention also relates to a genetically modified prokaryotic host cell, wherein the host cell is genetically modified such that (1) the activity of the fructose-6-phosphate converting enzyme, which is at the normal level in the unmodified host cell, is reduced or depressed; (2) at least one gene encoding an enzyme required for de novo GDP-fucose synthesis is overexpressed; (3) an exogenous gene encoding fucosyltransferase, preferably a gene encoding alpha-1,2-fucosyltransferase and/or alpha-1,3-fucosyltransferase, is expressed in the cell.

As mentioned for the above method, the host cell is preferably selected from an Escherichia coli strain, a Lactobacillus strain, or a Corynebacterium strain.

According to one embodiment of the host cell of the invention, the intracellular amount of fructose-6-phosphate is increased by (1) decreasing or inhibiting the activity of a fructose-6-phosphate converting enzyme selected from the group of phosphofructokinase, glucose-6-phosphate isomerase, fructose-6 α-phosphate aldolase, transketolases, eg tktA, tktB, or transaldolases, eg talA, talB, or (2) increased activity of fructose 1,6-bisphosphate phosphatase.

In a preferred embodiment, the genes encoding the enzymes required for de novo synthesis of GDP-fucose are overexpressed.

In yet another preferred embodiment, the exogenous genes encoding at least one fucosyltransferase are genes encoding alpha-1,2-fucosyltransferases and/or alpha-1,3-fucosyltransferases and are selected from the wbgL gene from E. coli O126 or the fucT2 gene from Helicobacter pylori when referring to alpha-1,2-fucosyltransferases, and from genes from Akkermansia muciniphila, Bacteroides fragilis, Helicobacter pylori or Helicobacter hepaticus species when referring to alpha-1,3-fucosyltransferases.

In one embodiment, the host cell described above may have been further genetically modified (4) to express an exogenous gene encoding an efflux sugar transporter; and/or (5) for the purpose of expressing an exogenous gene encoding a bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase; and/or (6) for the purpose of inactivating or deleting genes encoding L-fucose isomerase and L-fuculose kinase; and/or (7) to inactivate or disrupt genes encoding UDP(uridine diphosphate)-glucose:undecaprenyl phosphate-glucose-1-phosphate transferase; and/or (8) to express exogenous lactose permease; and/or (9) to inactivate or delete endogenous beta-galactosidase genes; and/or (10) for the purpose of expressing an exogenously regulated gene encoding beta-galactosidase; and/or (11) for the purpose of expressing exogenous genes responsible for the metabolization of galactose; and/or for the purpose of expressing an exogenous gene encoding fructose-1,6-bisphosphate phosphatase.

The present invention also relates to the use of a genetically modified prokaryotic host cell of the invention for the production of a fucosylated oligosaccharide.

Other advantages follow from the description of the embodiments and the attached drawings.

Obviously, the above-mentioned features and features that are still to be explained below can be used not only in suitably specified combinations, but also in other combinations or alone, without deviating from the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are illustrated in the figures and explained in more detail in the following part of the description. The figures show:

Fig. 1. Schematic exemplary illustration of a genetically modified host cell to be used in the method of the invention;

Fig. 2. Results of analysis of supernatants from glycerol-containing cultures in which 2'-fucosyllactose-producing strains of E. coli were grown using high performance liquid chromatography (HPLC); And

Fig. 3. SEQ ID no. 1-7.

DETAILED DESCRIPTION OF FIGURES AND EMBODIMENTS

On FIG. 1 shows an exemplary illustration of a host cell of the invention to be used in the process of the invention, showing exemplary fermentation pathways for exemplary fucosylated oligosaccharides, 2'-fucosyllactose. On FIG. 1 shows a typical bacterial host cell genetically modified according to the invention to produce 2'-fucosyllactose.

As can be seen in FIG. 1, glycerol is usually used as the carbon source, while lactose is added externally. Lactose is transported into the host cell via a permease (eg LacY). Glycerol is transferred into the host cell by facilitated diffusion via GlpF. Within the prokaryotic host cell, glycerol is converted to glyceraldehyde-3-phosphate, which is converted to fructose-6-phosphate (1) preferentially by overexpression of the exogenous gene encoding Fbpase and (2) by inhibition of the reverse reaction through inactivation of phosphofructokinase A (PfkA). Due to the overexpression of exogenous enzymes necessary for the de novo synthesis of GDP-fucose, i.e. phosphomannomutase, ManB, mannose-1-phosphate-guanosyltransferase, ManC, GDP-mannose-4,6-dehydratase, Gmd, and GDP-L-fucose synthase, WcaG, GDP-L-fucose is produced.

In the next step, GDP-L-fucose is reacted with an alpha-1,2-fucosyltransferase, such as WbgL, with internalized lactose to produce 2-fucosyllactose, which is exported via an efflux carrier, such as TPYb, to the medium in which the host cell is cultured.

On FIG. 2 shows the results of analyzes of supernatants from glycerol-containing cultures in which 2'-fucosyllactose-producing E. coli strains were grown using HPLC.

On FIG. 2 shows the HPLC profile of a broth sample after fermentation of a 2'-fucosyllactose-producing strain carrying the yberc0001_9420 gene encoding a heterologous transporter (black) and the HPLC profile of a broth sample after fermentation of the same strain but deleting the yberc0001_9420 gene encoding a heterologous transporter. (grey colour). Both strains were fermented for 111 h at 28°C using glycerol as a carbon and energy source.

Example 1

Construction of E. coli strain BL21(DE3) for 2'-fucosyllactose production

Using E. coli BL21(DE3) as the original host strain, a strain was designed to produce 2'-fucosyllactose by whole cell biosynthesis. The genomic construction of such a strain included the events of destruction and deletion of genes and the integration of heterologous genes.

Because 2'-fucosyllactose is synthesized from lactose, which is used in bacterial culture, and from GDP-L-fucose, which is produced by living cells, first a wild-type copy of the lacZ gene encoding endogenous β-galactosidase was inactivated by mutagenesis using mismatched oligonucleotides ( see Ellis et al., "High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides", Proc. Natl. Acad. Sci. USA, 98: 6742-6746 (2001)). Using the same method, the gene encoding arabinose isomerase, araA, was destroyed.

The lacZΩ fragment gene was introduced under the control of the temperature-sensitive transcriptional repressor cI857. The lacZα fragment gene is expressed under the control of the PgbA promoter from E. coli BL21 (DE3) in this strain, resulting in this strain LacZ ⁺ .

Genomic deletions were performed using phage-mediated λ Red-dependent recombination according to the method of Datsenko and Warner (see "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", Proc. Natl. Acad. Sci. USA , 97: 6640-6645 (2000)). The fucI and fucK genes encoding L-fucose isomerase and L-fuculose kinase, respectively, were deleted to prevent the degradation of L-fucose. The wzxC-wcaJ genes were also deleted. In all likelihood, the wcaJ gene encodes UDP-glucose:undecaprenyl-phosphate-glucose-1-phosphate transferase, which catalyzes the first step in the synthesis of colanoic acid (see Stevenson et al., "Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colonic acid", J. Bacteriol., 178: 4885-4893, (1996)); in the case of HDF-fucose, the production of colanoic acid will be carried out using a reaction involving fucosyl transferase activity.

Integration into the genome of heterologous genes was performed by transposition. Large gene clusters were integrated into the genome mediated by a hyperactive C9 mutant form of mariner Himar1 transposase (see Lampe et al., "Hyperactive transposase mutants of the Himar1 mariner transposon", Proc. Natl. Acad. Sci. USA, 96: 11428- 11433 (1999)), whose gene was inserted into the pEcomar plasmid under the transcriptional control of the P _ara promoter. To enhance de novo synthesis of GDP-fucose, genes encoding phosphomannomutase (manB), mannose-1-phosphate guanosyltransferase (manC), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose synthase (wcaG) from E. coli K12 DH5α was overexpressed in E. coli BL21(DE3); the manCB operon was placed under the control of the constitutive promoter P _tet , the gmd, wcaG operon is transcribed starting from the constitutive P _T5 promoter. Transposon cassette <P _tet -manCB-P _T5 -gmd, wcaG-FRT-dhfr-FRT> (SEQ ID No. 1), including the dihydrofolate reductase (dhfr) gene for acquiring resistance to trimethoprim, flanked by inverted terminal repeats specifically recognized mariner-like element of Himar1 transposase was inserted into the E. coli genome from pEcomar C9-manCB-gmd, wcaG-dhfr.

For chromosomal integration of single genes, transposase EZ-Tn5™ (Epicentre, USA) was used. To generate EZ-Tn5 transposomes, the gene of interest along with the FRT site flanked by an antibiotic resistance cassette was amplified along with primers that carried 19 base pair (b.p.) recognition sites at both sites - mosaic ends (5 '-CTGTCTCTTATACACATCT (SEQ ID No. 8)) for EZ-Tn5 transposase. Using the EZ-Tn5™ transposase, the gene encoding the lactose importer LacY from E. coli K12 TG1 (accession number ABN72583), the 2-fucosyl transferase gene, wbgL, from E. coli: O126 (accession number ADN43847) and the yberc0001_9420 gene encoding efflux sugar transporter from the major transporter superfamily from Yersinia bercovieri, ATCC (American Type Structure Collection) 43970 (accession number EEQ08298) using the appropriate integration cassettes: <P _tet -lacY-FRT-aadA-FRT> (SEQ ID No. 2), < P _tet -wbgLcoFRT-neoFRT> (SEQ ID No. 3) and P _tet -yberc0001_9420coFRT-cat-FRT> (SEQ ID No. 4) to give a strain. The wbgL and yberc0001_9420 genes were synthesized and codon-optimized (simultaneously) by the GenScript Cooperation (USA). After successful integration of the lacY gene, the resistance gene was removed from streptomycin-resistant clones by FLP recombinase encoded by 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 BL21(DE3) lacks a functional gal operon, a naturally regulated copy of the galETKM operon from E. coli K was integrated into strain B via EZ transposition using the integration cassette <P _gal -galE-galT-galK-galM> (SEQ ID No. 5). The integrated samples were taken from MacConkey agar containing 1% galactose as red colonies. The resulting strain is able to metabolize the monosaccharides glucose and galactose formed during the hydrolysis of lactose.

Example 2

Examination of enhanced export of 2'-fucosyllactose by the efflux sugar transporter from Yersinia bercovieri, ATCC 43970

yberc0001_9420 gene knockout

To demonstrate the functionality of the heterologous sugar transporter from Yersinia bercovieri, ATCC 43970, the yberc0001_9420 gene was deleted from E. coli strain BL21(DE3) lacZ ^- , araA ^- , fucI ^- , fucK ^- , wcaJ ^- , which contained chromosomal integrations of the manB, manC, gmd genes. , wcaG, lacY, wbgL; and yberc0001_9420 were deleted by heterologous recombination according to Datsenko and Wanner (2000; supra) using the aacC1 gentamicin resistance cassette from plasmid pBBR-MCS5 (Kovach, Elzer et al. 1995, "Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes", Gene, 166, 175-176), which was inserted into the yberc0001_9420 gene, resulting in the Δyberc0001_9420 strain.

Culture conditions for obtaining 2'-fucosyllactose

The E. coli strain BL21 (DE3) carrying the yberc0001_9420 gene encoding the heterologous transporter and the Δyberc0001_9420 strain were cultured at 28°C in 3 L fermenters (New Brunswick, Edison, USA) starting from 800 ml of mineral salt medium containing NH ₄ H ₂ PO ₄ - 7 g / l, K ₂ HPO ₄ - 7 g / l, KOH - 2 g / l, citric acid - 0.3 g / l, MgSO ₄ × 7H ₂ O - 2 g / l and CaCl ₂ × 6H ₂ O - 0.015 g / l, supplemented with a solution of trace elements at the rate of 1 ml / l (containing ammonium-iron (III) citrate - 54.4 g / l, MnCl ₂ × 4H ₂ O - 9.8 g / l, CoCl ₂ × 6H ₂ O - 1.6 g / l, CuCl ₂ × 2H ₂ O - 1 g / l, H ₃ BO ₃ - 1.9 g / l, ZnSO ₄ × 7H ₂ O - 9 g / l, Na ₂ MoO ₄ × 2H ₂ O -1.1 g / l, Na ₂ SeO ₃ - 1.5 g / l, NiSO ₄ × 6H ₂ O - 1.5 g / l), containing 1.5% glycerol as a carbon source and the antibiotics trimethoprim (10 µg/ml) and kanamycin (15 µg/ml). Cultivation was started using 2.5% (v/v) inoculum from a preculture growing in the same glycerol-containing medium. Lactose as an acceptor in the fucosyltransferase reaction was added over a period of seven hours, resulting in a concentration of 30 mM in the culture mixture, starting at an optical density at 660 nm (OD _{660 nm} ) of about 10. The lactose content was then manually adjusted to maintain an excess of acceptor molecules; glycerol was added continuously.

Analysis of culture supernatant and detection of 2'-fucosyllactose by HPLC

Analysis by high performance liquid chromatography (HPLC) was performed using a refractive index detector (RID-10A) (Shimadzu, Germany) and a ReproSil Carbohydrate, 5 μm (250 mm × 4.6 mm) column (Dr. Maisch GmbH, Germany) attached to HPLC system (Shimadzu, Germany). Elution was performed isocratically using acetonitrile:H ₂ O (68/32 (v/v)) as eluent at 35° C. and a flow rate of 1.4 ml/min. 20 µl of the sample was loaded onto the column. The concentration of 2'-fucosyllactose was calculated from the standard curve. For this purpose, 10% (v/v) 100 mM sucrose solution was added to HPLC samples as an internal standard, after which they were filtered (pore size 0.22 μm) and purified by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex ).

Detection of 2'-fucosyllactose in culture supernatants of E. coli BL21(DE3)

After 111 hours of fermentation at 28°C in a medium with mineral salts and glycerol as a carbon source, HPLC detected 2'-fucosyllactose at a concentration of 73 mM (35.6 g/l) and 25 mM (12.2 g/l) in culture supernatant of strains with and without the yberc0001_9420 gene encoding the transporter, see FIG. 2: FIG. 2 shows the HPLC profile for broth after fermentation of a 2'-fucosyllactose-producing strain carrying the yberc0001_9420 gene encoding a heterologous transporter (black) and the HPLC profile for broth after fermentation of the same strain but deleting the yberc0001_9420 gene encoding a heterologous transporter. (grey colour). Deletion in the strain of the yberc0001_9420 gene encoding a heterologous sugar transporter reduces the detectable amount of 2'-fucosyllactose in the supernatant. This suggests that the carrier protein does enhance 2'-fucosyllactose production by more rapidly transporting this trisaccharide out of the cell, since the genetic environment, with the exception of the yberc0001_9420 gene, is identical in both strains. In addition, a lower cell density was achieved in the case of cells lacking the 2'-fucosyllactose transporter, probably due to osmotic stress caused by significant accumulation of sugar within the cells. As shown in FIG. 2, the amount of 2',3-difucosyllactose found in the culture of the strain Δyberc0001_9420 is approximately two times greater than in the broth with the original strain. Increased production of 2',3-difucosyl lactose when L-fucose is transferred to 2'-fucosyl lactose in a fucosyl transferase-catalyzed reaction also indicates higher intracellular concentrations of the 2'-fucosyl lactose acceptor molecule in the yberc0001_9420 gene knockout strain compared to the yberc0001_9420 overexpressing strain. .

On FIG. 2 with lighter lines, i.e. gray lines show the supernatant of E. coli BL21(DE3) strain Δyberc0001_9420 Δyberc0001_9420, black lines show the supernatant from a culture containing the yberc0001_9420 gene strain E. coli BL21(DE3). Samples were taken after 111 h of fermentation at 28°C in a medium with mineral salts using glycerol as a carbon source.

Example 3

Obtaining 2'-fucosyllactose Enzymatically

The fermentation processes were carried out in fermenters with a capacity of 3 l at 30°C and at pH 7.0; the pH value was adjusted by titration with 25% ammonia solution. The strain described in Example 2 was cultured in the mineral salt medium described in Example 2 using glycerol as a carbon and energy source. The contents of the fermenter with an initial volume of 1 l were inoculated with a preculture cultivated in the same medium. After spending 2% of the glycerol contained in this portion, glycerol (60%, v/v) was fed continuously. Lactose at a concentration of 0.66 M was added in three portions (at intervals of one hour) of 10 ml each when an OD _{600 nm} value of 6 was reached. After that, lactose was continuously fed to maintain the concentration of lactose in the fermenter at least 10 mm . After 86 hours of cultivation, a final concentration of 2'-fucosyllactose of 91.3 mM (44.6 g/l) was reached. By raising the temperature to 42° C., the β-galactosidase gene is expressed, and lactose and its cleavage products, glucose and galactose, are metabolized by the 2'-fucosyllactose-producing strain.

Example 4

HPLC Analysis of Culture Supernatant

HPLC analysis was performed using a refractive index detector (RID-10A) (Shimadzu, Germany) and an XBridge amide sorbent column, 3.5 μm (250×4.6 mm) from Waters (Eschborn, Germany) connected to an HPLC system ( Shimadzu, Germany). Elution was performed isocratically using 30% A:50% (v/v) ACN (acetonitrile) in deionized water (ddH ₂ O) with 0.1% (v/v) NH as eluent. ₄ OH, and 70% B: 80% (v/v) ACN in ddH ₂ O, with 0.1% (v/v) NH ₄ OH, at 35°C and at a flow rate of 1.4 ml/min. 10 μl of the sample was applied to the column and the concentration of 2'-fucosyllactose was calculated from the standard curve. To this end, 10% (v/v) 100 mM sucrose solution was added to HPLC samples as an internal standard before filtration (pore size 0.22 μm) and purification by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). By-products such as L-fucose, 3-fucosyllactose, 2',3-difucosyllactose and fucosylgalactose were also detected using the same assay conditions.

Example 5

Improvement of 2'-fucosyllactose-producing strain using metabolic engineering

Further improvement regarding the synthesis of 2'-fucosyllactose using an E. coli strain was achieved by deleting the pfkA gene encoding phosphofructokinase A. When E. coli is cultivated on a gluconeogenic substrate such as glycerol, phosphorylation of fructose-6-phosphate by PfkA is a typical reaction with a large consumption of ATP, and in addition to this there is competition with ManA for the substrate. The pfkA gene was deleted using heterologous recombination according to Datsenko and Wanner (2000, supra), using a gentamicin resistance cassette (aacCl) flanked by lox71/66 sites (see Lambert, Bongers 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 successful deletion of the pfkA gene, the antibiotic resistance gene was removed from the E. coli genome using Cre recombinase (see Abremski, Hoess et al., 1983, "Studies on the properties of P1 site-specific recombination: evidence for topological-L unlinked products following recombination", Cell, 32, 1301-1311), which was cloned under the control of the P _ara promoter in the backbone of plasmid pKD46 (see Datsenko and Wanner, 2000).

For various fucosyltransferases, in addition to the transferase activity, GDP-L-fucosehydrolase activity was demonstrated. In addition, such hydrolytic activity has been shown for wbgL, an alpha-1,2-fucosyltransferase used for the synthesis of 2'-fucosyllactose (see EP 3050973 A1). To conserve free L-fucose to produce 2'-fucosyllactose and eliminate contaminating L-fucose from the culture broth, the fkp gene encoding the bifunctional L-fucokinase/L-fucose-1-phosphate guanylyl transferase from Bacteroides fragilis is under the transcriptional control of the P _tet promoter. , together with the aacCl gene flanked by lox71/66, was integrated into the chromosome of the strain described in example 1 by transposition using the EZ-Tn5™ transposase; <Ptet-fkp-lox-aacC1-lox> (SEQ ID 6). After successful integration, the gentamicin resistance gene was removed from the genome as described above.

Example 6

Optimized fermentation process for obtaining 2'-fucosyllactose

Using an optimized medium with mineral salts, which contains KH ₂ PO ₄ - 3 g/l, K ₂ HPO ₄ - 12 g/l, (NH ₄ ) ₂ SO ₄ - 5 g/l, citric acid - 0.3 g/l l, MgSO ₄ × 7H ₂ O - 2 g / l, NaCl - 0.1 g / l and CaCl ₂ × 6H ₂ O - 0.015 g / l, together with a solution of trace elements at the rate of 1 ml / l (containing ammonium citrate- iron (III) - 54.4 g / l, MnCl ₂ × 4H ₂ O - 9.8 g / l, CoCl ₂ × 6H ₂ O - 1.6 g / l, CuCl ₂ × 2H ₂ O - 1 g / l, H ₃ BO ₃ - 1.9 g / l, ZnSO ₄ × 7H ₂ O - 9 g / l, Na ₂ MoO ₄ × 2H ₂ O - 1.1 g / l, Na ₂ SeO ₃ - 1.5 g/l, NiSO ₄ ×6H ₂ O - 1.5 g/l), and 2% glycerol as a carbon source, the E. coli strain described in example 5 was cultivated in a 3 l fermenter at 33°C. The pH value was maintained at 7.0 by titration with 25% ammonia solution. The contents of the fermenter were inoculated with a preculture with OD _600nm 0.1 growing in the same medium. Lactose was added when an OD _{600 nm} value of 5 was observed for the culture, resulting in a concentration of 30 mM. Throughout the fermentation process, the lactose concentration was maintained at 20-30 mM, which was monitored using HPLC assays. Feeding with glycerol (60%, v/v) was started after consuming the glycerol in the fermenter contents at flow rates of 4.5 ml/l/h for 20 hours, after which feeding was carried out for 33 hours at 5.7 ml/l /h and for 18 hours at 7.1 ml/l/h for a period of 18 hours (feed rates refer to initial volume). A total of 106.5 g/l (217 mM) 2'-fucosyllactose was reached after 93 hours.

Example 7

Construction of an improved 2'-fucosyllactose-producing strain by stimulating metabolic activity

To enhance the flow of the metabolizable carbon source glycerol through the gluconeogenic pathway from triose phosphates to fructose-6-phosphate to fuel the biosynthesis of GDP-L-fucose, the genes encoding fructose-1,6-biphosphate aldolase (fbaB) and the heterologous fructose-1 gene, 6-bisphosphate-phosphatases (fbpase) from Pisum sativum were overexpressed in the strain described in Example 5. The fbaB gene from E. coli BL21 (DE3) was fused to the P _tet promoter. FBPase activity from P. sativum chloroplasts is allosterically regulated by a disulfide-dithiol exchange mediated by reduction involving thioredoxins. Replacing the cysteine residue 153 with a series results in a constitutively active enzyme. The gene encoding the P. sativum chloroplast FBPase (accession number AAD10213), codon-optimized for expression in E. coli, with a hexahistidine tag at the N-terminus, and modified to encode the C153S variant of the enzyme, was purchased from Genescript. The fbpase gene is transcribed from the T7 promoter. The <P _tet -fbaB-P _T7 -His ₆ -fbpase-lox-aacC1-lox> cassette (SEQ ID No. 7) was used for EZ-Tn5™ transposase mediated integration into the host strain. After removing the gentamicin resistance gene from the E. coli genome, this strain was used to obtain 2'-fucosyllactose.

Example 8

Obtaining 2'-fucosyllactose at a concentration of 150 g/l by the enzymatic method

A strain producing 2'-fucosyllactose genetically modified as described in Example 7 was cultured in the same medium at 33°C as described in Example 5. In addition, a portion of 60 mM lactose in 2% was added initially to the fermentation medium. th glycerin. A continuous feed of lactose at a lactose concentration of 0.66M was started at an OD _600nm value of about 10. In addition, the addition of lactose was carried out using a 1M concentrated solution. The lactose concentration was maintained at a value of approximately 30 mm. After the end of the batch feeding phase, as indicated by an increase in the level of dissolved oxygen, feeding with glycerol (60%, v/v) was started at a flow rate of 6.9 ml/l/h for 37 hours (relative to the initial volume). Thereafter, the feed was reduced to 9.4 ml/l/h over 19 hours and then increased again to 7.3 ml/l/h over 19 hours. 93 hours after seeding the fermenter, a 2'-fucosyllactose concentration of 150.2 g/L was reached.

Example 9

Obtaining 3-fucosyllactose from glycerol

Using E. coli BL21 (DE3) with lacZ ΔwcaJ ΔfucIK with chromosomal integration of genes encoding enzymes for de novo GDP-fucose synthesis (ManB, ManC, Gmd, WcaG), a 3-fucosyllactose-producing strain was constructed.

The gene encoding alpha-1,3-fucosyltransferase from Bacteroides fragilis (EP 2439264 A1), together with the gene encoding the SetA efflux sugar transporter from E. coli (US 2014/0120611 A1) and the gene conferring gentamicin resistance, were integrated into genome of E. coli. Fermentation of the strain to produce 3-fucosyllactose was carried out under the conditions described in Example 6. Glycerol feeding was started after the end of the batch feeding phase at a feeding rate of 7.4 ml/l/h (relative to the initial volume). Lactose was added to the culture mixture to a concentration of 33 mM when an OD _{600 nm} of 30 was reached. Lactose was added throughout the process to maintain a concentration of at least 10 mM in the supernatant. After 88 hours, the process was stopped at a concentration of 3-fucosyllactose in the supernatant of 30 g/L.

--->

SEQUENCE LIST

<110> JENNEWEIN BIOTECHNOLOGY GMBH

<120> An improved method for obtaining fucosylated

oligosaccharides

<130> 2827P112EP

<140>EP 16 196 486.1

<141> 2016-10-29

<160> 8

<170>PatentIn version 3.5

<210> 1

<211> 6783

<212> DNA

<213> Artificial sequence

<220>

<223> Transposon cassette

<400> 1

gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt tattttacca 60

ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct agaaataatt 120

ttgtttaact ttaagaagga gatatacaat ttcgtcgaca cacaggaaac atattaaaaa 180

ttaaaacctg caggagtttg aaggagatag aaccatggcg cagtcgaaac tctatccagt 240

tgtgatggca ggtggctccg gtagccgctt atggccgctt tcccgcgtac tttatcccaa 300

gcagttttta tgcctgaaag gcgatctcac catgctgcaa accaccatct gccgcctgaa 360

cggcgtggag tgcgaaagcc cggtggtgat ttgcaatgag cagcaccgct ttattgtcgc 420

ggaacagctg cgtcaactga acaaacttac cgagaacatt attctcgaac cggcagggcg 480

aaacacggca cctgccattg cgctggcggc gctggcggca aaacgtcata gcccggagag 540

cgacccgtta atgctggtat tggcggcgga tcatgtgatt gccgatgaag acgcgttccg 600

tgccgccgtg cgtaatgcca tgccatatgc cgaagcgggc aagctggtga ccttcggcat 660

tgtgccggat ctaccagaaa ccggttatgg ctatattcgt cgcggtgaag tgtctgcggg 720

tgagcaggat atggtggcct ttgaagtggc gcagtttgtc gaaaaaccga atctggaaac 780

840

cgccggacgc tatctcgaag aactgaaaaa atatcgcccg gatatcctcg atgcctgtga 900

aaaagcgatg agcgccgtcg atccggatct caattttatt cgcgtggatg aagaagcgtt 960

tctcgcctgc cgggaagagt cggtggatta cgcggtcatg gaacgtacgg cagatgctgt 1020

tgtggtgccg atggatgcgg gctggagcga tgttggctcc tggtcttcat tatgggagat 1080

cagcgcccac accgccgagg gcaacgtttg ccacggcgat gtgattaatc acaaaactga 1140

aaacagctat gtgtatgctg aatctggcct ggtcaccacc gtcggggtga aagatctggt 1200

agtggtgcag accaaagatg cggtgctgat tgccgaccgt aacgcggtac aggatgtgaa 1260

aaaagtggtc gagcagatca aagccgatgg tcgccatgag catcgggtgc atcgcgaagt 1320

gtatcgtccg tggggcaaat atgactctat cgacgcgggc gaccgctacc aggtgaaacg 1380

catcaccgtg aaaccgggcg agggcttgtc ggtacagatg caccatcacc gcgcggaaca 1440

ctgggtggtt gtcgcgggaa cggcaaaagt caccattgat ggtgatatca aactgcttgg 1500

1560

aattccgctc gatttaattg aagtgcgctc cggctcttat ctcgaagagg atgatgtggt 1620

gcgtttcgcg gatcgctacg gacgggtgta aacgtcgcat caggcaatga atgcgaaacc 1680

gcggtgtaaa taacgacaaa aataaaattg gccgcttcgg tcagggccaa ctattgcctg 1740

1800

ttaggcgaag aactgaatga agatatcgcc tggcgcattg gtcgcgccta tggcgaattt 1860

ctcaaaccga aaaccattgt gttaggcggt gatgtccgcc tcaccagcga aaccttaaaa 1920

ctggcgctgg cgaaaggttt acaggatgcg ggcgttgacg tgctggatat tggtatgtcc 1980

ggcaccgaag agatctattt cgccacgttc catctcggcg tggatggcgg cattgaagtt 2040

accgccagcc ataatccgat ggattataac ggcatgaagc tggttcgcga gggggctcgc 2100

ccgatcagcg gagataccgg actgcgcgac gtccagcgtc tggctgaagc caacgacttt 2160

cctcccgtcg atgaaaccaa acgcggtcgc tatcagcaaa tcaacctgcg tgacgcttac 2220

gttgatcacc tgttcggtta tatcaatgtc aaaaacctca cgccgctcaa gctggtgatc 2280

aactccggga acggcgcagc gggtccggtg gtggacgcca ttgaagcccg ctttaaagcc 2340

ctcggcgcgc ccgtggaatt aatcaaagtg cacaacacgc cggacggcaa tttccccaac 2400

ggtattccta acccactact gccggaatgc cgcgacgaca cccgcaatgc ggtcatcaaa 2460

cacggcgcgg atatgggcat tgcttttgat ggcgattttg accgctgttt cctgtttgac 2520

gaaaaagggc agtttattga gggctactac attgtcggcc tgttggcaga agcattcctc 2580

gaaaaaaatc ccggcgcgaa gatcatccac gatccacgtc tctcctggaa caccgttgat 2640

gtggtgactg ccgcaggtgg cacgccggta atgtcgaaaa ccggacacgc ctttattaaa 2700

gaacgtatgc gcaaggaaga cgccatctat ggtggcgaaa tgagcgccca ccattacttc 2760

cgtgatttcg cttactgcga cagcggcatg atcccgtggc tgctggtcgc cgaactggtg 2820

tgcctgaaag ataaaacgct gggcgaactg gtacgcgacc ggatggcggc gtttccggca 2880

agcggtgaga tcaacagcaa actggcgcaa cccgttgagg cgattaaccg cgtggaacag 2940

cattttagcc gtgaggcgct ggcggtggat cgcaccgatg gcatcagcat gacctttgcc 3000

gactggcgct ttaacctgcg cacctccaat accgaaccgg tggtgcgcct gaatgtggaa 3060

tcgcgcggtg atgtgccgct gatggaagcg cgaacgcgaa ctctgctgac gttgctgaac 3120

gagtaaaaac cagtgcaaga atcataaaaa 3180

3240

ttttgtgagc ggataacaat tccccatctt agtatattag ttaagtataa ataccgcg 3300

gaggacgaag gagatagaac catgtcaaaa gtcgctctca tcaccggtgt aaccggacaa 3360

gacggttctt acctggcaga gtttctgctg gaaaaaggtt acgaggtgca tggtattaag 3420

3480 cgtcgcgcat cgtcattcaa caccgagcgc

tgcaacccga aattccatct gcattatggc gacctgagtg atacctctaa cctgacgcgc 3540

attttgcgtg aagtacagcc ggatgaagtg tacaacctgg gcgcaatgag ccacgttgcg 3600

gtctcttttg agtcaccaga atataccgct gacgtcgacg cgatgggtac gctgcgcctg 3660

ctggaggcga tccgcttcct cggtctggaa aagaaaactc gtttctatca ggcttccacc 3720

tctgaactgt atggtctggt gcaggaaatt ccgcagaaag agaccacgcc gttctacccg 3780

cgatctccgt atgcggtcgc caaactgtac gcctactgga tcaccgttaa ctaccgtgaa 3840

tcctacggca tgtacgcctg taacggaatt ctcttcaacc atgaatcccc gcgccgcggc 3900

gaaaccttcg ttacccgcaa aatcacccgc gcaatcgcca acatcgccca ggggctggag 3960

tcgtgcctgt acctcggcaa tatggattcc ctgcgtgact ggggccacgc caaagactac 4020

gtaaaaatgc agtggatgat gctgcagcag gaacagccgg aagatttcgt tatcgcgacc 4080

ggcgttcagt actccgtgcg tcagttcgtg gaaatggcgg cagcacagct gggcatcaaa 4140

ctgcgctttg aaggcacggg cgttgaagag aagggcattg tggtttccgt caccgggcat 4200

gacgcgccgg gcgttaaacc gggtgatgtg attatcgctg ttgacccgcg ttacttccgt 4260

ccggctgaag ttgaaacgct gctcggcgac ccgaccaaag cgcacgaaaa actgggctgg 4320

4380

gcgaaaaaac actctctgct gaaatctcac ggctacgacg tggcgatcgc gctggagtca 4440

taagcatgag taaacaacga gtttttattg ctggtcatcg cgggatggtc ggttccgcca 4500

tcaggcggca gctcgaacag cgcggtgatg tggaactggt attacgcacc cgcgacgagc 4560

tgaacctgct ggacagccgc gccgtgcatg atttctttgc cagcgaacgt attgaccagg 4620

tctatctggc ggcggcgaaa gtgggcggca ttgttgccaa caacacctat ccggcggatt 4680

tcatctacca gaacatgatg attgagagca acatcattca cgccgcgcat cagaacgacg 4740

tgaacaaact gctgtttctc ggatcgtcct gcatctaccc gaaactggca aaacagccga 4800

tggcagaaag cgagttgttg cagggcacgc tggagccgac taacgagcct tatgctattg 4860

ccaaaatcgc cgggatcaaa ctgtgcgaat catacaaccg ccagtacgga cgcgattacc 4920

gctcagtcat gccgaccaac ctgtacgggc cacacgacaa cttccacccg agtaattcgc 4980

atgtgatccc agcattgctg cgtcgcttcc acgaggcgac ggcacagaat gcgccggacg 5040

tggtggtatg gggcagcggt acaccgatgc gcgaatttct gcacgtcgat gatatggcgg 5100

cggcgagcat tcatgtcatg gagctggcgc atgaagtctg gctggagaac acccagccga 5160

tgttgtcgca cattaacgtc ggcacgggcg ttgactgcac tatccgcgag ctggcgcaaa 5220

ccatcgccaa agtggtgggt tacaaaggcc gggtggtttt tgatgccagc aaaccggatg 5280

gcacgccgcg caaactgctg gatgtgacgc gcctgcatca gcttggctgg tatcacgaaa 5340

tctcactgga agcggggctt gccagcactt accagtggtt ccttgagaat caagaccgct 5400

ttcgggggggg gagctaacgc gccatttaaa tcaacctcag cggtcatagc tgtttcctgt 5460

gactgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 5520

gctgaaacca atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac 5580

tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg 5640

aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgggatcc 5700

aggccggcct gttaacgaat taatcttccg cggcggtatc gataagcttg atatcgaatt 5760

ccgaagttcc tattctctag aaagtatagg aacttcaggt ctgaagagga gtttacgtcc 5820

agccaagcta gcttggctgc aggtcgtcga aattctaccg ggtaggggag gcgcttttcc 5880

caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg 5940

gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg 6000

gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc 6060

agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga 6120

tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc 6180

6240

ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca 6300

cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga 6360

cctgcagcct gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 6420

ggtgaggaac taaaccatgg gtcaaagtag cgatgaagcc aacgctcccg ttgcaggggca 6480

gtttgcgctt cccctgagtg ccacctttgg cttaggggat cgcgtacgca agaaatctgg 6540

tgccgcttgg cagggtcaag tcgtcggttg gtattgcaca aaactcactc ctgaaggcta 6600

tgcggtcgag tccgaatccc acccaggctc agtgcaaatt tatcctgtgg ctgcacttga 6660

acgtgtggcc taatgagggg atcaattctc tgatcagaag ttcctattct 6720

ctagaaagta taggaacttc gatggcgcct catccctgaa gccaataggg ataacagggt 6780

aat 6783

<210> 2

<211> 2851

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 2

tggccagatg attaattcct aatttttgtt gacactctat cattgataga gttatttttac 60

cactccctat cagtgataga gaaaagtgaa atgaatagtt cgacaaaaat ctagaaataa 120

ttttgtttaa ctttaagaag gagatataca aatgtactat ttaaaaaaca caaacttttg 180

240

cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta ttatttttgc 300

cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt ctgacaaact 360

cgggctgcgc aaatacctgc tgtggattat taccggcatg ttagtgatgt ttgcgccgtt 420

ctttattttt atcttcgggc cactgttaca atacaacatt ttagtaggat cgattgttgg 480

tggtatttat ctaggctttt gttttaacgc cggtgcgcca gcagtagagg catttattga 540

gaaagtcagc cgtcgcagta atttcgaatt tggtcgcgcg cggatgtttg gctgtgttgg 600

ctgggcgctg tgtgcctcga ttgtcggcat catgttcacc atcaataatc agtttgtttt 660

ctggctgggc tctggctgtg cactcatcct cgccgtttta ctctttttcg ccaaaacgga 720

tgcgccctct tctgccacgg ttgccaatgc ggtaggtgcc aaccattcgg catttagcct 780

taagctggca ctggaactgt tcagacagcc aaaactgtgg tttttgtcac tgtatgttat 840

tggcgtttcc tgcacctacg atgtttttga ccaacagttt gctaatttct ttacttcgtt 900

ctttgctacc ggtgaacagg gtacgcgggt atttggctac gtaacgacaa tgggcgaatt 960

acttaacgcc tcgattatgt tctttgcgcc actgatcatt aatcgcatcg gtgggaaaaa 1020

cgccctgctg ctggctggca ctattatgtc tgtacgtatt attggctcat cgttcgccac 1080

ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg tttgaagtac cgttcctgct 1140

ggtggggctgc tttaaatata ttaccagcca gtttgaagtg cgtttttcag cgacgattta 1200

1260

1320

gggcttcacc ttaatttccg tgttcacgct tagcggcccc ggcccgcttt ccctgctgcg 1380

tcgtcaggtg aatgaagtcg ctgggagcta agcggccgcg tcgacacgca aaaaggccat 1440

ccgtcaggat ggccttctgc ttaatttgat gcctggcagt ttatggcggg cgtcctgccc 1500

1560

tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag 1620

cctttcgttt tatttgatgc ctggcagttc cctactctcg catggggaga ccccacacta 1680

ccatcatgta tgaatatcct ccttagttcc tattccgaag ttcctattct ctagaaagta 1740

taggaacttc ggcgcgtcct acctgtgaca cgcgtgccgc agtctcacgc ccggagcgta 1800

1860

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgaggga 1920

agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca 1980

tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa 2040

gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg 2100

gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct 2160

ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc 2220

taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga 2280

gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt 2340

tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt 2400

tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga 2460

gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc 2520

gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt 2580

catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc 2640

agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa 2700

ataatgtcta acaattcgtt caagccgagg ggccgcaaga tccggccacg atgacccggt 2760

cgtcgggtac cggcagggcg gggcgtaagg cgcgccattt aaatgaagtt cctattccga 2820

agttcctatt ctctagaaag tataggaact t 2851

<210> 3

<211> 2858

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 3

ggccagatga ttaattccta atttttgttg acactctatc attgatagag ttattttacc 60

actccctatc agtgatagag aaaagtgaaa tgaaatagttc gacaaaaatc tagaaataat 120

tttgtttaac tttaagaagg agatatacaa atgggcagca ttattcgtct gcagggtggt 180

ctgggtaatc agctgtttca gtttagcttt ggttatgccc tgagcaaaat taatggtaca 240

ccgctgtatt tcgacattag ccattatgcc gaaaacgatg atcatggtgg ttatcgtctg 300

aataatctgc agattccggga agaatatctg cagtattata ccccgaaaat taataatatt 360

420

tgcaacgaat ttcatgccta tggctacgat tttgaatata ttgcccagaa atggaaaagc 480

aaaaaataca ttggctactg gcagagcgaa cacttttttc ataaacatat tctggacctg 540

aaagaatttt ttattccgaa aaatgtgagc gaacaggcaa atctgctggc agcaaaaatt 600

ctggaaagcc agagcagcct gagcattcat attcgtcgtg gcgattatat taaaaacaaa 660

accgcaaccc tgacacatgg tgtttgtagc ctggaatatt ataaaaaagc cctgaacaaa 720

atccgcgatc tggcaatgat tcgtgatgtg tttatcttta gcgacgatat cttctggtgc 780

aaagaaaata ttgaaaccct gctgagcaaa aaatataata ttttattatag cgaagatctg 840

agccaagaag aggatctgtg gctgatgagc ctggcaaatc atcatattat tgccaaatagc 900

agctttagtt ggtggggtgc atatctgggt agcagcgcaa gccagattgt tatttatccg 960

accccgtggt atgatattac cccgaaaaac acctatatcc cgattgtgaa ccattggatc 1020

aacgttgata aacatagcag ctgctaagcg gccgcgtcga cacgcaaaaa ggccatccgt 1080

caggatggcc ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca 1140

ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg tcctactcag 1200

gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 1260

tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc acactaccat 1320

catgtatgaa tatcctcctt agttcctatt ccgaagttcc tattctctag aaagtatagg 1380

aacttcggcg cgtcctacct gtgacacgcg tcaagatccc ctcacgctgc cgcaagcact 1440

cagggcgcaa gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt 1500

gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa 1560

agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat 1620

ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct 1680

gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat 1740

ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 1800

gttctccggc cgcttggggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 1860

gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 1920

agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc 1980

tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 2040

actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 2100

ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 2160

cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 2220

ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 2280

tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg 2340

atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 2400

gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 2460

aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 2520

attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg 2580

gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc 2640

cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 2700

ccagcgcggg gatctcatgc tggagttctt cgcccacccc agcttcaaaa gcgctctcgg 2760

taccggcagg gcggggcgta aggcgcgcca tttaaatgaa gttcctattc cgaagttcct 2820

attctctaga aagtatagga acttcgaagc agctccag 2858

<210> 4

<211> 2631

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 4

ggccagatga ttaattccta atttttgttg acactctatc attgatagag ttattttacc 60

actccctatc agtgatagag aaaagtgaaa tgaaatagttc gacaaaaatc tagaaataat 120

tttgtttaac tttaagaagg agatatacaa atgaagtcgg cactgacctt ttcccgtcgc 180

atcaatccgg tgtttctggc gttctttgtc gttgcttttc tgagcggtat cgcaggcgca 240

ctgcaggctc cgaccctgag tctgtttctg tccacggaag tgaaagttcg tccgctgtgg 300

gttggtctgt tctataccgt caacgcaatc gctggcatta cggttagctt tatcctggcg 360

aaacgttcag attcgcgcgg tgaccgtcgc aagctgatta tggtgtgcta tctgatggcg 420

gttggcaact gtctgctgtt tgccttcaat cgtgattacc tgaccctgat cacggcaggt 480

gtgctgctgg cgagcgttgc caacaccgca atgccgcaga ttttcgcgct ggcccgtgaa 540

tatgccgaca gctctgcacg cgaagtggtt atgtttagtt ccatcatgcg cgctcaactg 600

agtctggcat gggtgattgg tccgccgctg tcctttatgc tggcgctgaa ttacggtttt 660

accctgatgt tctcaatcgc ggccggcatt ttcgttctgt cggccctggt cgtgtggttt 720

atcctgccga gtgtcccgcg tgcagaaccg gttgtcgatg caccggtggt tgtccagggt 780

tcactgttcg cagacaaaaa cgttctgctg ctgtttatcg cgtcgatgct gatgtggacc 840

tgcaatacga tgtatattat cgatatgccg ctgtacatta ccgcaagcct gggtctgccg 900

gaacgtctgg ctggtctgct gatgggtacc gcagctggcc tggaaattcc gatcatgctg 960

ctggcgggtt attctgtgcg ttactttggc aaacgcaaga ttatgctgtt cgctgttctg 1020

gcgggtgtcc tgttttatac cggcctggtt ctgtttaaat tcaagacggc cctgatgctg 1080

ctgcagatct ttaacgcaat tttcatcggt attgtggctg gcattggtat gctgtacttc 1140

caagatctga tgccgggtcg tgcaggtgca gcaaccacgc tgtttaccaa tagcatctct 1200

acgggtgtca ttctggcagg cgtgctgcaa ggcggtctga ccgaaacgtg gggccatgac 1260

agcgtctatg tgatggcgat ggtcctgtct attctggccc tgattatctg tgcacgtgtg 1320

1380

aaaaggccat ccgtcaggat ggccttctgc ttaatttgat gcctggcagt ttatggcggg 1440

cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga 1500

tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct 1560

1620

ccccacacta ccatcatgta tgaatatcct ccttagttcc tattccgaag ttcctattct 1680

ctagaaagta taggaacttc ggcgcgtcct acctgtgacg gaagatcact tcgcagaata 1740

aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt ggcgaaaatg 1800

agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc 1860

1920

aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc 1980

atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt 2040

tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc 2100

ccgcctgatg aatgctcatc cggaattacg tatggcaatg aaagacggtg agctggtgat 2160

atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc 2220

2280

ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt 2340

cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga 2400

2460

gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagatg 2520

cttaatgaat acaacagtac tgcgatgagt ggcagggcgg ggcgtaaggc gcgccattta 2580

aatgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt c 2631

<210> 5

<211> 4259

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 5

ttactcagca ataaactgat attccgtcag gctggaatac tcttcgccag gacgcaggaa 60

gcagtccggt tgcggccatt cagggtggtt cgggctgtcc ggtagaaact cgctttccag 120

agccagccct tgccagtcgg cgtaaggttc ggttccccgc gacggtgtgc cgccgaggaa 180

gttgccggag tagaattgca gagccggagc ggtggtgtag accttcagct gcaatttttc 240

atctgctgac cagacatgcg ccgccacttt cttgccatcg cctttggcct gtaacaagaa 300

tgcgtgatcg taacctttca ctttgcgctg atcgtcgtcg gcaagaaact cactggcgat 360

gattttggcg ctgcggaaat caaaagacgt tccggcgaca gatttcaggc cgtcgtgcgg 420

aatgccgcct tcatcaaccg gcagatattc gtccgccaga atctgcaact tgtgattgcg 480

cacgtcagac tgctcgccgt caagattgaa atagacgtga ttagtcatat tcaccgggca 540

600

ttgcaccgtc gcgccgagat tacccgggaa gccctgatca ccatcatctg aactcagggc 660

aaacagcacc tgacgatcgt tctggttcac aatctgccag cgacgtttgt cgaacccttc 720

cggcccgccg tgcagctggt taacgccctg acttggcgaa agcgtcacgg tttcaccgtc 780

aaaggtataa cggctattgg cgatacggtt ggcataacga ccaatagagg cccccagaaa 840

cgcggcctga tcctgatagc attccgggct ggcacagccg agcagcgcct cgcggacgct 900

gccatcggaa agcggaatac gggcggaaag taaagtcgca ccccagtcca tcagcgtgac 960

taccatccct gcgttgttac gcaaagttaa cagtcggtac ggctgaccat cgggtgccag 1020

tgcgggagtt tcgttcagca ctgtcctgct ccttgtgatg gtttacaaac gtaaaaagtc 1080

tctttaatac ctgtttttgc ttcatattgt tcagcgacag cttgctgtac ggcaggcacc 1140

agctcttccg ggatcagcgc gacgatacag ccgccaaatc cgccgccggt catgcgtacg 1200

1260

gtgatttcga aatcatcgcg catagaggca tgagactccg ccatcaactc gcccatacgt 1320

ttcaggtcgc cttgctccag cgcgctggca gcttcaacgg tgcgggcgtt ttcagtcagt 1380

atatgacgca cgcgttttgc cacgatcggg tccagttcat gcgcaacagc gttgaactct 1440

tcaatggtga catcacgcag ggctggctgc tggaagaaac gcgcaccggt ttcgcactgt 1500

tcacgacggg tgttgtattc gctgccaacc agggtacgtt tgaagttact gttgatgatg 1560

acgacagcca cacctttgggg catggaaact gctttggtcc ccagtgagcg gcaatcgatc 1620

agcaaggcat gatctttctt gccgagcgcg gaaattagct gatccatgat cccgcagtta 1680

cagcctacaa actggttttc tgcttcctga ccgttaagcg cgatttgtgc gccgtccagc 1740

ggcagatgat aaagctgctg caatacggtt ccgaccgcga cttccagtga agcggaagaa 1800

cttaacccgg caccctgcgg cacattgccg ctgatcacca tgtccacgcc gccgaagctg 1860

ttgttacgca gttgcagatg tttcaccacg ccacgaacgt agttagccca ttgatagttt 1920

tcatgtgcga caatgggcgc atcgagggaa aactcgtcga gctgattttc ataatcggct 1980

gccatcacgc gaactttacg gtcatcgcgt ggtgcacaac tgatcacggt ttgataatca 2040

atcgcgcagg gcagaacgaa accgtcgttg tagtcggtgt gttcaccaat caaattcacg 2100

cggccaggcg cctgaatggt gtgagtggca gggtagccaa atgcgttggc aaacagagat 2160

tgtgtttttt ctttcagact catttcttac actccggatt cgcgaaaatg gatatcgctg 2220

actgcgcgca aacgctctgc tgcctgttct gcggtcaggt ctcgctgggt ctctgccagc 2280

atttcataac caaccataaa tttacgtacg gtggcggagc gcagcagagg cggataaaag 2340

tgcgcgtgca gctgccagtg ttgattctct tcgccattaa atggcgcgcc gtgccagccc 2400

atagagtagg ggaaggagca ctggaagagg ttgtcataac gactggtcag ctttttcaac 2460

gccagcgcca gatcgctgcg ctgggcgtcg gtcaaatcgg tgatccgtaa aacgtgggct 2520

ttgggcagca gtagcgtttc gaacggccag gcagcccagt aaggcacgac ggctaaccag 2580

tgttcggttt cgacaacggt acggctaccg tctgccagct cgcgctgaac ataatccacc 2640

agcattggtg atttctgttc ggcaaaatat tctttttgca ggcggtcttc gcgctcagct 2700

2760

atcgccgcgc ctttgttttc aaaaacctgc acccatgggt acgttttccc cagttctgcg 2820

gtttgctcct gccaggtttt gacgatttcc gtcaatgctg caacgctgag ctctggcagc 2880

gttttactgt gatccggtga aaagcagatc acccggctgg tgccgcgcgc gctctggcaa 2940

cgcatcagcg gatcgtgact ttctggcgca tctggcgtgt cagacatcaa agccgcaaag 3000

tcattagtga aaacgtaagt cccggtgtaa tcggggtttt tatcgcctgt cacccgcaca 3060

ttacctgcgc agaggaagca atctggatcg tgcgcaggta acacctgttt ggctggcgtt 3120

3180

gtgagcgggt tgtagcggcg atgtggatga tcaacgggat taaattgcgt catggtcgtt 3240

ccttaatcgg gatatccctg tggatggcgt gactgccagt gccaggtgtc ctgcgccatt 3300

tcatcgagtg tgcgcgttac gcgccagttc agttcacggt cggctttgct ggcgtccgcc 3360

3420

caggctttgc tgaaggcatt aaccacgtcc agcacgctgt tgcctacgcc agcgccgagg 3480

ttgtagatgt gtacgcctgg cttgttcgcc agtttttcca tcgccacgac gtgaccgtcc 3540

gccagatcca ttacgtggat gtaatcgcgt acgccagtac catcttcggt cggataatcg 3600

ttaccaaaaa tcgccagcga gtcgcgacgg cctacagcaa cctgggcgat gtatggcatc 3660

aggttattcg gaatgccttg cggatcttcg cccatatcgc ccgacggatg cgcgccaacc 3720

gggttgaagt agcgcagcag ggcaatgctc cagtccggct gggctttttg cagatcggtg 3780

3840

3900

ataaagtttt tgacgttagc ggcgcgcatg gcgctaatca ggcgcagagt gccgttgaca 3960

ttgttgtcgt aatattccag cggtttttgt accgattcgc ccacggcttt cagcccggcg 4020

aagtggatca cggtgtcgat agcgtgatcg tgcaggatct cggtcatcaa cgcttcgtta 4080

4140

acgctgcgct tactgttaca gaggttatca agaatgatga catcatgacc gttttgcagt 4200

aattgcacac aggtatgact tccaatgtaa ccgctaccac cggtaaccag aactctcat 4259

<210> 6

<211> 4223

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 6

tggccagatg attaattcct aatttttgtt gacactctat cattgataga gttatttttac 60

cactccctat cagtgataga gaaaagtgaa atgaatagtt cgacaaaaat ctagaaataa 120

ttttgtttaa ctttaagaag gagatataca aatgcaaaaa ctactatctt taccgtccaa 180

tctggttcag tcttttcatg aactggagag ggtgaatcgt accgattggt tttgtacttc 240

cgacccggta ggtaagaaac ttggttccgg tggtggaaca tcctggctgc ttgaagaatg 300

ttataatgaa tattcagatg gtgctacttt tggagagtgg cttgaaaaag aaaaaagaat 360

tcttcttcat gcgggtgggc aaagccgtcg tttacccggc tatgcacctt ctggaaagat 420

tctcactccg gttcctgtgt tccggtggga gagagggcaa catctgggac aaaatctgct 480

ttctctgcaa cttcccctat atgaaaaaat catgtctttg gctccggata aactccatac 540

actgattgcg agtggtgatg tctatattcg ttcggagaaa cctttgcaga gtattcccga 600

agcggatgtg gtttgttatg gactgtgggt agatccgtct ctggctaccc atcatggcgt 660

gtttgcttcc gatcgcaaac atcccgaaca actcgacttt atgcttcaga agccttcgtt 720

ggcagaattg gaatctttat cgaagaccca tttgttcctg atggacatcg gtatatggct 780

tttgagtgac cgtgccgtag aaatcttgat gaaacgttct cataaagaaa gctctgaaga 840

actaaagtat tatgatcttt attccgattt tggattagct ttgggaactc atccccgtat 900

tgaagacgaa gaggtcaata cgctatccgt tgctattctg cctttgccgg gaggagagtt 960

ctatcattac gggaccagta aagaactgat ttcttcaact ctttccgtac agaataaggt 1020

ttacgatcag cgtcgtatca tgcaccgtaa agtaaagccc aatccggcta tgtttgtcca 1080

aaatgctgtc gtgcggatac ctctttgtgc cgagaatgct gatttatgga tcgagaacag 1140

1200

1260

gggctttgtt gcccgtccat acggtctgga cgatgttttc aaaggagatt tgagagattc 1320

1380

tacagatttg aaaggacgta cggacgattt acaggcagtt tccgtattcc ctatggttaa 1440

ttctgtagaa gagttgggat tggtgttgag gtggatgttg tccgaacccg aactggagga 1500

aggaaagaat atctggttac gttccgaaca ttttttctgcg gacgaaattt cggcaggtgc 1560

caatctgaag cgtttgtatg cacaacgtga agagttcaga aaaggaaact ggaaagcatt 1620

ggccgttaat catgaaaaaa gtgtttttta tcaacttgat ttggccgatg cagctgaaga 1680

ttttgtacgt cttggtttgg atatgcctga attattgcct gaggatgctc tgcagatgtc 1740

acgcatccat aaccggatgt tgcgtgcgcg tattttgaaa ttagacggga aagattatcg 1800

tcgggaagaa caggctgctt ttgatttgct tcgtgacggc ttgctggacg ggatcagtaa 1860

tcgtaagagt accccaaaat tggatgtata ttccgatcag attgtttggg gacgtagccc 1920

cgtgcgcatc gatatggcag gtggatggac cgatactcct ccttattcac tttattcggg 1980

aggaaatgtg gtgaatctag ccattgagtt gaacggacaa cctcccttac aggtctatgt 2040

gaagccgtgt aaagacttcc atatcgtcct gcgttctatc gatatgggtg ctatggaaat 2100

2160

gaaagccgct ctgtcattgg caggctttgc acctgcgttt tctgctgtat cttatgcttc 2220

attagaggaa cagcttaaag atttcggtgc aggtattgaa gtgactttat tggctgctat 2280

tcctgccggt tccggtttgg gcaccagttc cattctggct tctaccgtac ttggtgccat 2340

taacgatttc tgtggtttag cctgggataa aaatgagatt tgtcaacgta ctcttgttct 2400

tgaacaattg ctgactaccg gaggtggatg gcaggatcag tatggaggtg tgttgcaggg 2460

tgtgaagctt cttcagaccg aggccggctt tgctcaaagt ccattggtgc gttggctacc 2520

cgatcattta tttacgcatc ctgaatacaa agactgtcac ttgctttatt ataccggtat 2580

aactcgtacg gcaaaaggga tcttggcaga aatagtcagt tccatgttcc tcaattcatc 2640

gttgcatctc aatttacttt cggaaatgaa ggcgcatgca ttggatatga atgaagctat 2700

acagcgtgga agttttgttg agtttggccg tttggtagga aaaacctggg aacaaaacaa 2760

2820

ttataccttg ggatataaat tgccgggagc cggtggtggc gggtacttat atatggtagc 2880

gaaagatccg caagctgctg ttcgtattcg taagatactg acagaaaacg ctccgaatcc 2940

3000

actgaaacca atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac 3060

tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg 3120

aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgggatcc 3180

aggccggcct gttaagacgg ccagtgaatt cgagctcggt acctaccgtt cgtataatgt 3240

atgctatacg aagttatcga gctctagaga atgatcccct cattaggcca cacgttcaag 3300

tgcagcgcac accgtggaaa cggatgaagg cacgaaccca gttgacataa gcctgttcgg 3360

ttcgtaaact gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg 3420

3480

tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga 3540

tgttatggag cagcaacgat gttacgcagc agcaacgatg ttacgcagca gggcagtcgc 3600

cctaaaacaa agttaggtgg ctcaagtatg ggcatcattc gcacatgtag gctcggccct 3660

gaccaagtca aatccatgcg ggctgctctt gatcttttcg gtcgtgagtt cggagacgta 3720

gccacctact cccaacatca gccggactcc gattacctcg ggaacttgct ccgtagtaag 3780

acattcatcg cgcttgctgc cttcgaccaa gaagcggttg ttggcgctct cgcggcttac 3840

gttctgccca ggtttgagca gccgcgtagt gagatctata tctatgatct cgcagtctcc 3900

ggcgagcacc ggaggcaggg cattgccacc gcgctcatca atctcctcaa gcatgaggcc 3960

aacgcgcttg gtgcttatgt gatctacgtg caagcagatt acggtgacga tcccgcagtg 4020

gctctctata caaagttggg catacgggaa gaagtgatgc actttgatat cgacccaagt 4080

accgccacct aacaattcgt tcaagccgag atcgtagaat ttcgacgacc tgcagccaag 4140

cataacttcg tataatgtat gctatacgaa cggtaggatc ctctagagtc gacctgcagg 4200

catgagatgt gtataagaga cag 4223

<210> 7

<211> 3792

<212> DNA

<213> Artificial sequence

<220>

<223> Integration cassette

<400> 7

gggaattgat tctggtacca aatgagtcga ccggccagat gattaattcc taatttttgt 60

tgacactcta tcattgatag agttatttta ccactcccta tcagtgatag agaaaagtga 120

aatgaatagt tcgacaaaaa tctagaaata attttgttta actttaagaa ggagatatac 180

240

acgggagcat agtaatgaca gatattgcac agttgcttgg caaagacgcc gacaaccttt 300

tacagcaccg ttgtatgact attccttctg accagcttta tctccccggga catgactacg 360

tagaccgcgt gatgattgac aataatcgcc cgccagcggt gttacgtaat atgcagacgt 420

tgtacaacac tgggcgtctg gctggcacag gatatctttc tattctgccg gttgaccagg 480

gcgttgagca ctctgccgga gcttcatttg ctgctaaccc gctctacttt gacccgaaaa 540

acattgttga actggcgatc gaagcgggct gtaactgtgt ggcatcaact tacggcgtgt 600

tggcgtcggt atcgcggcgc tatgcgcatc gcattccatt cctcgtcaaa cttaatcaca 660

acgagacgct aagttacccg aacacctacg atcaaacgct gtatgccagc gtggagcagg 720

ccttcaacat gggcgcggtg gcggttggtg cgactatcta ttttggttcg gaagagtcac 780

gtcgccagat tgaagaaatt tctgcggctt ttgaacgtgc gcacgagctg ggcatggtga 840

cagtgctgtg ggcctatttg cgtaactccg cctttaagaa agatggcgtt gattaccatg 900

tttccgccga cctgaccggt caggcaaacc atctggcggc gaccataggt gcagatatcg 960

tcaaacaaaa aatggcggaa aataacggcg gctataaagc aattaattac ggttataccg 1020

acgatcgcgt gtacagcaag ttaaccagcg aaaacccgat tgatctggtg cgttatcagt 1080

tagctaactg ctatatgggc cgggccgggt tgataaactc cggcggtgct gcaggcggtg 1140

aaactgacct cagcgatgca gtgcgtactg cggttatcaa caaacgcgct ggcggaatgg 1200

ggctgattct tggacgtaag gcgttcaaga aatcgatggc tgacggcgtg aaactgatta 1260

acgccgtgca ggatgtttat ctcgatagca aaattactat cgcctaagag gatcgagatc 1320

tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1380

ctctagaaat aattttgttt aactttaaga aggagatata ccatggggcca tcatcatcat 1440

catcatcatc atcatcacag cagcggccat atcgaaggtc gtcatatggc ggtgaaagaa 1500

gcgaccagcg agaccaagaa gcgtagcggt tacgagatca ttaccctgac cagctggctg 1560

ctgcaacaag aacagaaggg tatcattgac gcggaactga ccatcgttct gagcagcatt 1620

agcatggcgt gcaaacagat cgcgagcctg gtgcaacgtg cgaacattag caacctgacc 1680

ggtacccaag gcgcggttaa catccagggt gaagaccaaa agaaactgga tgttattagc 1740

aacgaggtgt tcagcaactg cctgcgtagc agcggtcgta ccggcatcat tgcgagcgag 1800

gaagaggacg tggcggttgc ggtggaagag agctacagcg gtaactatat cgtggttttt 1860

gacccgctgg atggcagcag caacctggat gcggctgtga gcaccggtag catcttcggc 1920

atttacagcc cgaacgacga gagcctgccg gattttggtg acgatagcga cgataacacc 1980

ctgggcaccg aagagcaacg ttgcatcgtt aacgtgtgcc aaccggggtag caacctgctg 2040

gcggcgggct actgcatgta tagcagcagc gttgcgttcg tgctgaccat tggcaagggc 2100

gttttcgtgt ttaccctgga cccgctgtac ggtgaattcg tgctgaccca ggagaacctg 2160

2220

gaaaacctga agaaatatat cgacgatctg aaggaaccgg gtccgagcgg taaaccgtac 2280

agcgcgcgtt atatcggtag cctggttggc gacttccacc gtaccctgct gtacggtggc 2340

atttacggtt atccgcgtga taagaaaagc aagaacggca aactgcgtct gctgtatgaa 2400

tgcgcgccga tgagctttat tgttgagcag gcgggtggca aaggtagcga cggccaccag 2460

cgtgtgctgg atatccaacc gaccgaaatt caccagcgtg ttccgctgta cattggtagc 2520

accgaagagg ttgaaaaagt tgaaaagtat ctggcgtaat cgagtctggt aaagaaaccg 2580

ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct 2640

aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 2700

2760

tagcggcgca ttaagcgcgg cgggtggacg gccagtgaat tcgagctcgg tacctaccgt 2820

tcgtataatg tatgctatac gaagttatcg agctctagag aatgatcccc tcattaggcc 2880

acacgttcaa gtgcagcgca caccgtggaa acggatgaag gcacgaaccc agttgacata 2940

agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg cgctcacgca actggtccag 3000

aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga 3060

3120

3180

agggcagtcg ccctaaaaca aagttaggtg gctcaagtat gggcatcatt cgcacatgta 3240

ggctcggccc tgaccaagtc aaatccatgc gggctgctct tgatcttttc ggtcgtgagt 3300

tcggagacgt agccacctac tcccaacatc agccggactc cgattacctc gggaacttgc 3360

tccgtagtaa gacattcatc gcgcttgctg ccttcgacca agaagcggtt gttggcgctc 3420

tcgcggctta cgttctgccc aggtttgagc agccgcgtag tgagatctat atctatgatc 3480

tcgcagtctc cggcgagcac cggaggcagg gcattgccac cgcgctcatc aatctcctca 3540

agcatgaggc caacgcgctt ggtgcttatg tgatctacgt gcaagcagat tacggtgacg 3600

atcccgcagt ggctctctat acaaagttgg gcatacggga agaagtgatg cactttgata 3660

tcgacccaag taccgccacc taacaattcg ttcaagccga gatcgtagaa tttcgacgac 3720

ctgcagccaa gcataacttc gtataatgta tgctatacga acggtaggat cctctagagt 3780

cgacctgcag gc 3792

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 8

ctgtctctta tacacatct 19

<---

Claims (18)

1. A method for producing 2'-fucosyllactose using a genetically modified prokaryotic host cell, comprising the steps: - providing a prokaryotic host cell genetically modified such that at least (1) the activity of a fructose-6-phosphate converting enzyme that is at the normal level in the unmodified host cell is reduced or suppressed, wherein said fructose converting enzyme -6-phosphate, is phosphofructokinase, and/or by increasing the activity of fructose-1,6-bisphosphate-phosphatase; (2) at least one gene encoding an enzyme required for de novo synthesis of GDP-fucose is overexpressed in the host cell, wherein said at least one gene encoding an enzyme required for de novo synthesis of GDP-fucose is a gene , encoding the ManA enzyme catalyzing the isomerization of fructose-6-phosphate to mannose-6-phosphate, a gene encoding phosphomannomutase, a gene encoding mannose-1-phosphate guanosyltransferase, a gene encoding GDP-mannose-4,6-dehydratase, or a gene encoding GDP-L-fucose synthase; (3) an exogenous gene encoding alpha-1,2-fucosyltransferase is expressed in the host cell; and (4) the gene encoding fructose 1,6-biphosphate aldolase is overexpressed; - culturing and/or growing said genetically modified host cell in a culture medium containing a source of carbon and/or energy, which is glycerol; And - ensuring the supply of lactose to the culture medium; thereby obtaining a fucosylated oligosaccharide which can be recovered from the medium in which the host cell is cultured. 2. The method of claim 1 wherein the host cell is selected from the group consisting of bacterial host cells. 3. The method according to claim 1 or 2, where the amount of fructose-6-phosphate in the cell increases as a result of an increase in the activity of fructose-1,6-bisphosphate-phosphatase. 4. The method according to any one of paragraphs. 1-3, where the gene encoding phosphomannomutase is manB , the gene encoding mannose-1-phosphate guanosyltransferase is manC , the gene encoding GDP-mannose-4,6-dehydratase is gmd , and the gene encoding GDP-L-fucose synthase, is wca G. 5. The method of claim 4, wherein the gene encoding alpha-1,2-fucosyltransferase is selected from the group consisting of the wbgL gene from E. coli O126 or the fucT2 gene from Helicobacter pylori . 6. The method according to any one of paragraphs. 1-5 wherein the host cell is further genetically modified to express a gene encoding a protein that exports or facilitates the export of the desired fucosylated oligosaccharide to the culture medium. 7. The method according to any one of paragraphs. 1-6, where endogenous or exogenous lactose import permease is overexpressed. 8. The method according to any one of paragraphs. 1-7, where the genes that are modified in the host cell or that the host cell is modified are endogenous or exogenous genes. 9. The method according to any one of paragraphs. 1-8, wherein at least one of the genes that are modified in the host cell or that are modified in the host cell is overexpressed by endogenous or exogenous induction, or in a constitutive manner. 10. The method of claim 9, wherein the gene encoding the protein that exports or promotes the export of the desired 2'-fucosyllactose is a gene encoding an efflux sugar transporter. 11. The method according to any one of paragraphs. 3-10, wherein fructose 1,6 bisphosphate phosphatase is encoded by a gene that is a functionally active variant of the fructose 1,6 bisphosphate phosphatase ( fbpase ) gene from Pisum sativum as present in SEQ ID NO. 7. 12. The method according to any one of paragraphs. 7-10, where the lactose permease is LacY from E. coli . 13. The method according to any one of paragraphs. 1-12, where the provision of lactose is carried out by adding lactose from the beginning of cultivation at a concentration of at least 5 mm. 14. The method according to any one of paragraphs. 1-13, where the provision of lactose is carried out by adding lactose to the culture medium in such a concentration that during the phase of formation of the product during cultivation, a lactose concentration of at least 5 mm is reached. 15. The method according to any one of paragraphs. 1-14, where the host cells are cultured for at least 60 hours.

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