Fermentation production of carbohydrates by using microbial cells of mixed raw materials

Abstract

This application discloses genetically engineered microbial cells for producing target carbohydrates, wherein the microbial cells have increased intracellular availability of at least one phosphate sugar and produce the target carbohydrates when cultured in a medium containing a mixed monosaccharide feedstock as the primary carbon and energy source. This application also discloses a fermentation method for producing the target carbohydrates by culturing the genetically engineered microbial cells in the presence of a mixed monosaccharide feedstock as the primary carbon and energy source.

Description

[0001] This invention relates to the fermentation production of a target sugar. This application discloses microbial cells capable of producing the target sugar, wherein the microbial cells utilize a mixed monosaccharide feedstock as the primary carbon and energy source during fermentation. This application also discloses a method for producing the target sugar using said microbial cells. Background Technology

[0002] Human milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals, and trace elements. The most abundant component of human milk is carbohydrates. These carbohydrates can be further divided into (i) lactose and (ii) oligosaccharides (human milk oligosaccharides, HMOs). While the disaccharide lactose (galactose-β1,4-glucose) is used as an energy source by the infant, oligosaccharides are not metabolized by the infant.

[0003] Oligosaccharides account for up to one-tenth of the total carbohydrate fraction and consist of approximately 150 different oligosaccharides. The presence and concentration of these complex oligosaccharides are unique to humans and therefore cannot be found in large quantities in the milk of other mammals, including dairy animals.

[0004] The most important human milk oligosaccharides are 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), which together account for up to one-third of the total HMO fraction. Other important HMOs are lact-N-tetrasaccharide (LNT), lact-N-neotetrasaccharide (LNnT), and lact-N-fucopentose I (LNFP-I). In addition to these neutral oligosaccharides, acidic HMOs can also be found in human milk, such as 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose, sialyl-lact-N-tetrasaccharide, and disialialyl-lact-N-tetrasaccharide.

[0005] It is noteworthy that the vast majority of HMOs contain a galactose-β1,4-glucose moiety at their reducing end, which is extended by adding a monosaccharide moiety (such as N-acetylglucosamine (GlcNAc) and / or fucose and / or galactose and / or N-acetylneuraminic acid (NeuNAc)). The structure of HMOs is closely related to epitopes of epithelial cell surface glycoconjugates (Lewis tissue blood group antigens, such as Lewis x (LeX)). The structural similarity between HMOs and epithelial epitopes explains the protective properties of HMOs against bacterial pathogens.

[0006] The presence of oligosaccharides in human milk has long been known, and their physiological functions have been the subject of medical research for decades. Specific functions have been identified for some of the more abundant oligosaccharides in human milk.

[0007] In addition to the local effects in the gut mentioned earlier, HMOs have also been shown to induce systemic effects in infants by entering the systemic circulation. Furthermore, the effects of HMOs on protein-carbohydrate interactions (e.g., selector protein-leukocyte binding) can modulate immune responses and reduce inflammatory responses. Moreover, there is a growing recognition that HMOs represent key substrates for infant microbiome development.

[0008] While the beneficial properties of various carbohydrates (especially prebiotic oligosaccharides) have been extensively studied in general, there is a great need for an efficient and cost-effective method for producing monosaccharides (e.g., L-fucose, N-acetylneuraminic acid), disaccharides (e.g., lact-N-biose), and oligosaccharides (e.g., 2'-FL, LNnT) due to the limited availability of their natural sources.

[0009] In an attempt to mass-produce functional carbohydrates, chemical pathways for some of these carbohydrates have been developed. However, these methods involve the use of several toxic chemicals, which increases the risk of contaminating the final product. To date, chemical synthesis has not been able to provide functional carbohydrates in sufficient quantities and quality for food applications.

[0010] To avoid the drawbacks associated with the chemical synthesis of human milk oligosaccharides, several enzymatic and fermentation methods have been developed for their production. Several fermentation methods have been developed for the production of carbohydrates such as L-fucose, N-acetylneuraminic acid, 2'-fucosyllactose, 3-fucosyllactose, lact-N-tetrasaccharide, lact-N-neotetrasaccharide, 3'-sialyllactose, and 6'-sialyllactose. These production methods typically utilize genetically engineered bacterial cells, such as recombinant Escherichia coli.

[0011] Typically, fermentation processes and biocatalytic reactions for producing HMOs are based on the exogenous addition of lactose as the initial acceptor substrate for the HMOs to be produced. In these methods, one or more monosaccharides are added to lactose (US 7,521,212 B1; Albertmannet al., (2001) Carbohydr. Res. 334(2) pp. 97-103). The addition of monosaccharides to lactose can be catalyzed by glycosyltransferases or glycosidases, using suitable activated monosaccharide substrates. Furthermore, other monosaccharides can be added to lactose via transglycosidase reactions.

[0012] Specifically, the fermentation production of HMOs has proven efficient because the metabolism of the microbial cells used provides the essential but difficult-to-synthesize nucleotide-activated monosaccharides. The biosynthetic pathways of nucleotide-activated monosaccharides typically originate from primary metabolism in the host cell at the glucose-6-phosphate or fructose-6-phosphate level. The biosynthesis of UDP-galactose (UDP-Gal) originates from glucose-6-phosphate, while the biosynthesis of GDP-fucose, UDP-N-acetylglucosamine, and CMP-N-acetylneuraminic acid originates from fructose-6-phosphate.

[0013] The efficient biosynthesis of nucleotide-activated monosaccharides in microbial host cells, and thus the production of desired carbohydrates via fermentation, clearly depends on a continuous supply of phosphate sugars such as glucose-6-phosphate and / or fructose-6-phosphate.

[0014] The main problem with these fermentation methods (which are typically based on microbial host cells consuming simple and inexpensive carbon and energy sources such as glycerol, glucose, and sucrose) is the limited availability of such phosphorylated / activated sugars in the host cells (e.g., due to competitive reactions), which greatly reduces the carbon flux of the product biosynthetic pathway, thereby reducing the productivity of these methods.

[0015] To overcome the aforementioned drawbacks, improved methods and approaches for producing HMOs have been developed. For example, WO 2012 / 007481 A2 discloses engineered organisms capable of producing sugars, activated sugars, nucleosides, glycosides, glycolipids, and glycoproteins, wherein the engineered organism expresses i) a gene encoding a carbohydrate hydrolase combined with a gene encoding a carbohydrate kinase, ii) a gene encoding a carbohydrate synthase, or iii) a gene encoding a carbohydrate phosphorylase, such that the organism is capable of cleaving disaccharides, oligosaccharides, polysaccharides, or mixtures thereof into activated sugars and glucose, and wherein the organism is further genetically modified such that at least one other gene besides any introduced gene of the organism is endowed with lower functionality or non-functionality, and wherein the other gene encodes enzymes and / or biocatalytic enzymes that convert the activated sugars into biomass. The engineered organism is capable of producing the desired carbohydrates while utilizing disaccharides such as sucrose, oligosaccharides, polysaccharides, or mixtures thereof.

[0016] However, a major drawback of using sucrose as the sole carbon and energy source in the engineered organisms to produce the desired compounds is the difficulty in sterilizing sucrose. The ideal sterilization method is heat sterilization. However, heat sterilization of sucrose results in a considerable degree of hydrolysis, negating the applicability of the method described in WO 2012 / 007481 A2.

[0017] As an alternative to heat sterilization, sterile filtration of the sucrose solution can be used; however, sterile filtration carries a high risk of contamination, particularly in industrial-scale fermentation, leading to the growth of unwanted bacterial cells.

[0018] However, due to its availability and low cost, sucrose represents the most attractive carbon source for biotechnology applications.

[0019] Therefore, there is a need for microbial cells for the fermentation production of desired carbohydrates, which, when cultured in the presence of inexpensive raw materials as the primary carbon and energy source, are capable of producing the desired carbohydrates, wherein the raw materials need not be hydrolyzed by the microbial cells for utilization by the cells' metabolism, and a method for producing the desired carbohydrates by fermentation of the microbial cells in the presence of the raw materials as the primary carbon and energy source.

[0020] The objective is achieved by providing genetically engineered microbial cells and a method for producing desired carbohydrates through fermentation; said microbial cells, when cultured on a mixed monosaccharide feedstock serving as the primary carbon and energy source, are capable of producing the desired carbohydrates, said mixed monosaccharide feedstock consisting of glucose and at least one other monosaccharide selected from fructose and galactose, and said microbial cells having increased intracellular availability of at least one phosphate sugar; said method comprising culturing said genetically engineered microbial cells in the presence of said mixed monosaccharide feedstock serving as the primary carbon and energy source. Summary of the Invention

[0021] According to a first aspect, this application provides genetically engineered microbial cells for producing target carbohydrates, wherein the microbial cells have increased intracellular availability of at least one phosphate sugar and are capable of producing target carbohydrates when cultured in the presence of a mixed monosaccharide feedstock as the primary carbon and energy source, wherein the mixed monosaccharide feedstock consists of glucose and at least one other monosaccharide selected from fructose and galactose.

[0022] According to the second aspect, this application provides the use of genetically engineered microbial cells as described herein for the production of desired carbohydrates, wherein the microbial cells are cultured in the presence of a monosaccharide mixture consisting of glucose and at least one other monosaccharide derived from fructose and galactose.

[0023] According to a third aspect, this application provides a method for fermenting to produce carbohydrates for a specific purpose, the method comprising the following steps:

[0024] a) Provide genetically engineered microbial cells capable of producing desired carbohydrates, wherein the microbial cells have increased intracellular availability of at least one phosphate sugar;

[0025] b) Culturing the genetically engineered microbial cells in a culture medium that allows the production of the desired carbohydrate, wherein the culture medium contains a mixed monosaccharide feedstock as the primary carbon and energy source, wherein the mixed monosaccharide feedstock comprises glucose and at least one other monosaccharide selected from fructose and galactose; and

[0026] c) Recover the target carbohydrates.

[0027] In a fourth aspect, this application provides the use of target carbohydrates produced by genetically engineered microbial cells and / or by the methods described herein in the preparation of pharmaceutical and / or nutritional compositions. Attached Figure Description

[0028] Figure 1 A schematic diagram of an exemplary wild-type microbial cell (e.g., Escherichia coli) is shown, illustrating the metabolic pathways that lead to and are derived from naturally occurring phosphate-fructose-1-phosphate and glucose-6-phosphate.

[0029] Figure 2 A schematic diagram of an exemplary genetically modified microbial cell of the present invention is shown, indicating a genetic modification that leads to increased intracellular glucose-6-phosphate availability.

[0030] Figure 3 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that lead to increased intracellular glucose-6-phosphate availability.

[0031] Figure 4 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, which indicates the genetic modifications that lead to increased intracellular glucose-6-phosphate availability.

[0032] Figure 5 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular glucose-6-phosphate availability and increased intracellular fructose-6-phosphate availability.

[0033] Figure 6 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular glucose-6-phosphate availability and increased intracellular fructose-6-phosphate availability.

[0034] Figure 7A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular glucose-6-phosphate availability and increased intracellular fructose-6-phosphate availability.

[0035] Figure 8 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular glucose-6-phosphate availability and increased intracellular fructose-6-phosphate availability.

[0036] Figure 9 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular glucose-6-phosphate availability and increased intracellular fructose-6-phosphate availability.

[0037] Figure 10 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular availability of glucose, glucose-6-phosphate, and fructose-6-phosphate.

[0038] Figure 11 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased intracellular mannose and / or mannose-6-phosphate availability.

[0039] Figure 12 A schematic diagram of another exemplary embodiment of the genetically engineered microbial cell of the present invention is shown, indicating genetic modifications that result in increased availability of intracellular galactose-1-phosphate and fructose-6-phosphate.

[0040] Figure 13 The growth characteristics of Escherichia coli strains during cultivation on glucose (A) or a mixed monosaccharide feed consisting of glucose and fructose (B) as the sole carbon and energy sources are shown. Detailed Implementation

[0041] According to the first aspect, genetically engineered microbial cells capable of producing a desired carbohydrate are provided. In another embodiment, the desired carbohydrate is a carbohydrate that is not naturally present in the wild-type progenitor cells of the genetically engineered microbial cells.

[0042] Compared to the intracellular availability of at least one phosphate sugar in the corresponding wild-type cells, the microbial cells have increased intracellular availability of at least one phosphate sugar. The genetically engineered microbial cells are capable of producing the target carbohydrate, and produce the target carbohydrate when cultured in a medium containing a mixed monosaccharide feedstock as the primary carbon and energy source for the microbial cells, wherein the mixed monosaccharide feedstock consists of glucose and at least one other monosaccharide selected from fructose and galactose.

[0043] Genetically engineered microbial cells are capable of producing target carbohydrates due to their genetic engineering. Therefore, genetically engineered microbial cells express one or more heterologous genes, wherein the activity of polypeptides encoded by said heterologous genes enables the microbial cell to synthesize the target carbohydrates.

[0044] As used herein, the term "functional gene" refers to a nucleic acid molecule containing a nucleotide sequence encoding a protein or polypeptide, and also containing a regulatory sequence operatively linked to the nucleotide sequence encoding the protein, such that the nucleotide sequence encoding the protein or polypeptide can be expressed in / by microbial cells having the functional gene. Thus, when cultured under conditions allowing for the expression of a functional gene, the functional gene is expressed, and the microbial cells expressing the functional gene typically contain a protein or polypeptide encoded by the protein-coding region of the functional gene. As used herein, the terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, include known analogs of natural nucleotides that hybridize with nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence includes its complementary sequence.

[0045] As used herein, the term "operably linked" refers to a functional link between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or a series of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression regulatory sequence influences the transcription and / or translation of the nucleic acid corresponding to the second sequence. Accordingly, the term "promoter" refers to a DNA sequence that typically precedes a gene in a DNA polymer and provides the site of initiation for transcription to mRNA. "Regulator" DNA sequences also typically precede a gene in a given DNA polymer and bind to proteins that determine the frequency (or rate) of transcription initiation. These sequences preceding a selected gene (or series of genes) in a functional DNA polymer are collectively referred to as "promoter / regulator" or "regulatory" DNA sequences, which work together to determine whether transcription (and eventual expression) of the gene occurs. DNA sequences following a gene in a DNA polymer and providing a termination signal for transcription to mRNA are called transcription "terminator" sequences.

[0046] The term "recombinant," when used herein to refer to bacterial host cells, means 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 "exogenous to the cell"). Recombinant cells may contain genes not present in the cell in its natural (non-recombinant) form. Recombinant cells may also contain genes present in the cell in its natural form, wherein said genes have been modified and artificially reintroduced into the cell. The term also covers cells containing modified nucleic acids that are endogenous to the cell, without removing said nucleic acids from the cell; such modifications include those obtained through gene substitution, site-specific mutations, and related techniques. Thus, a "recombinant polypeptide" is a polypeptide produced by recombinant cells. As used herein, a "heterologous sequence" or "heterologous nucleic acid" is a nucleic acid derived from a source other than a specific host cell (e.g., from a different species), or, if from the same source, modified in its natural form. Thus, a heterologous nucleic acid operatively linked to a promoter is from a source different from the source of the promoter, or, if from the same source, modified in its natural form. Heterologous sequences can be stably introduced (e.g., via transfection, transformation, conjugation, or transduction) into the genome of a host microbial cell, wherein the applicable techniques depend on the host cell into which the sequence is to be introduced. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0047] Therefore, "genetically engineered host cells" or "genetically engineered microbial cells" should be understood as bacterial or yeast cells that have been transformed or transfected, or bacterial or yeast cells that can be transformed or transfected by exogenous polynucleotide sequences.

[0048] Therefore, the nucleic acid sequences used in this invention may, for example, be contained in a vector that will be stably transformed / transfected or otherwise introduced into host microbial cells.

[0049] A variety of expression systems can be used to generate the peptides of the present invention. Such vectors include, in particular, chromosome-derived vectors, episome-derived vectors, and virus-derived vectors, such as those derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and combinations thereof, such as those derived from plasmids and bacteriophage genetic elements (e.g., granules and phage particles). Expression system constructs may include regulatory regions that regulate and induce expression. Generally, any system or vector suitable for maintaining, proliferating, or expressing polynucleotides and suitable for peptide synthesis in a host can be used for expression in this regard. Suitable DNA sequences can be inserted into the expression system using any of a variety of known and conventional techniques (e.g., those described above in Sambrook et al.).

[0050] Numerous patents and publications in the art relate to methods for "recombinant DNA" that isolate, synthesize, purify, and amplify genetic material for transformation of selected host organisms. Therefore, it is common knowledge to transform host organisms using "hybrid" viral or circular plasmid DNA containing selected exogenous (i.e., exogenous or "heterologous") DNA sequences. Procedures known in the art begin by generating a transformation vector by enzymatically digesting the circular viral or plasmid DNA to form a linear DNA strand. The selected exogenous DNA strand, typically containing a sequence encoding the desired protein product, is prepared in a linear form using the same / similar enzymes. The linear viral or plasmid DNA is incubated with the exogenous DNA in the presence of a ligase capable of performing the recovery process, forming a "hybridization" vector containing the selected exogenous DNA fragment "spliced" into the viral or circular DNA plasmid.

[0051] The term "nucleotide sequence encoding..." generally refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, and typically represents a portion of a gene encoding a polypeptide or protein. This term includes, but is not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions or a mixture of single-stranded, double-stranded, and triple-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA (which may be single-stranded or more typically double-stranded or triple-stranded regions, or a mixture of single-stranded and double-stranded regions). The term also covers polynucleotides comprising a single continuous or discontinuous region encoding a polypeptide (e.g., interrupted by an integrated phage, insertion, or edit) and may also comprise other regions encoding and / or non-coding sequences.

[0052] As used herein, the term "variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains the essential (enzymatic) properties of the reference polynucleotide or polypeptide. A typical polynucleotide variant has a nucleotide sequence that differs from another reference polynucleotide. Changes in the variant's nucleotide sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical polypeptide variant has an amino acid sequence that differs from another reference polypeptide. Typically, the differences are limited, making the sequences of the reference polypeptide and the variant very similar overall and identical in many regions. The differences in the amino acid sequences of the variant and the reference polypeptide can be any combination of one or more substitutions, additions, or deletions. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides can be naturally occurring, such as allelic variants, or can be variants known not to exist naturally. Non-natural variants of polynucleotides and polypeptides can be prepared by mutagenesis, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0053] Within the scope of this invention, those terms also include nucleic acid / polynucleotide and polypeptide polymorphism variants, alleles, mutants, and interspecies homologs whose amino acid sequences have greater than about 60% amino acid sequence identity with the polypeptide encoded by the wild-type protein, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater amino acid sequence identity, preferably in regions of at least about 25, 50, 100, 200, 500, 1000 or more amino acids.

[0054] Therefore, any “functional variant” of a gene / protein disclosed herein is intended to refer to a sequence variant of a gene / protein that retains the same or slightly lower activity as the gene or protein from which the fragments are derived.

[0055] The genetically engineered microbial cells possess at least one monosaccharide transporter for transporting at least one monosaccharide from the culture medium in which the microbial cells are cultured into their cytoplasm. The at least one monosaccharide transporter transports monosaccharides selected from glucose, fructose, and galactose.

[0056] Monosaccharides transported across the cell membrane must be phosphorylated to become available for cellular metabolism. Depending on the monosaccharide transporter that transports the monosaccharide across the cell membrane, the monosaccharide is either directly phosphorylated, i.e., when it is transferred into the cell, or subsequently phosphorylated by a suitable kinase. Transport of monosaccharides via phosphoenolpyruvate:sugar phosphoryltransferase-dependent transport (PEP-PTS) results in direct phosphorylation, while transport via phosphoenolpyruvate:sugar phosphoryltransferase-independent transport (non-PEP-PTS) requires subsequent phosphorylation by an intracellular kinase.

[0057] In an additional and / or alternative implementation, the microbial cell contains a glucose transport phosphotransferase system (PtsG). The glucose transport phosphotransferase system catalyzes the phosphorylation of incoming glucose, accompanied by its transmembrane transport.

[0058] The general mechanism of the Pts system is as follows: the phosphoryl group of phosphoenolpyruvate (PEP) is transferred to enzyme I (EI) via a signal transduction pathway, and enzyme I (EI) then transfers it to the phosphoryl carrier histidine protein (HPr). Phospho-HPr then transfers the phosphoryl group to a sugar-specific permease—a membrane-bound complex called enzyme 2 (EII)—which transports sugars into the cell. EII consists of at least three structurally distinct domains, IIA, IIB, and IIC. They can be fused together in a single polypeptide chain or exist as two or three interacting chains, formerly known as enzymes II (EII) and III (EIII).

[0059] The first domain (IIA or EIIA) carries the first permease-specific phosphorylation site—a histidine phosphorylated by phosphate-HPr. The second domain (IIB or EIIB) is phosphorylated by phosphate-IIA onto a cysteine ​​or histidine residue, depending on the sugar being transported. Finally, the phosphoryl group is transferred from the IIB domain to the sugar substrate, accompanied by sugar uptake handled by the IIC domain. The third domain (IIC or EIIC) forms the transport channel and the specific substrate binding site.

[0060] Therefore, the PtsG system obtains exogenous glucose and provides glucose-6-phosphate in microbial cells. Glucose-6-phosphate can be used in the UDP-galactose biosynthesis pathway and / or converted to fructose-6-phosphate, which in turn can be used to produce energy-rich triphosphates in central metabolism and / or, for example, in the biosynthesis of nucleotide-activated sugars (such as GDP-fucose).

[0061] In an additional and / or alternative embodiment, the genetically engineered microbial cells contain a fructose transporter protein for transporting fructose (Fru) from the culture medium into the cytoplasm of the microbial cells. A suitable fructose transporter protein for taking up free fructose is the isotype (PtsG-F) described as in Kornberg et al. PNAS 97:1808-1812 (2000).

[0062] The internalized fructose can then be phosphorylated by fructokinase (FrK) to provide fructose-6-phosphate (Fru-6-P). Fructose-6-phosphate can be used in the UDP-galactose biosynthesis pathway and / or other metabolic pathways, such as in central metabolism and / or in the biosynthesis of nucleotide-activated sugars (e.g., GDP-fucose) to produce high-energy triphosphates.

[0063] In an additional and / or alternative implementation, the genetically engineered microbial cell contains a fructose transport phosphotransferase system (PtsF). The fructose transport phosphotransferase system catalyzes the phosphorylation of incoming fructose, accompanied by its transmembrane transport.

[0064] Therefore, the PtsF system acquires exogenous fructose and provides fructose-1-phosphate in microbial cells. The PtsF system comprises the transmembrane protein FruA, phosphofructokinase 1-phosphate (FruK), and the diphosphotransferase protein FruB. Fructose is transported via FruA and FruB to provide fructose-1-phosphate in the cytoplasm. Fructose-1-phosphate can be further phosphorylated by phosphofructokinase (FruK) to produce fructose-1,6-bisphosphate, which can then be used by microbial cells to produce high-energy triphosphates in central metabolism.

[0065] Another suitable PtsF system comprises LevD, LevE, LevF, and LevG. LevD is the fructose-specific phosphotransferase IIA component. LevE is the fructose-specific phosphotransferase IIB component. LevF is the fructose permease IIC component, and LevG is the fructose permease IID component. The corresponding genes levD, levE, levF, and levG are known, for example, from Bacillus subtilis (strain 168). The PtsF system provides fructose-1-phosphate in the cell.

[0066] In an additional and / or alternative embodiment, the genetically engineered microbial cells possess a UDP-galactose biosynthetic pathway for the formation of intracellular UDP-galactose (UDP-Gal). UDP-galactose is required as a substrate for galactosyltransferases, wherein the activity of said galactosyltransferases can lead to the formation of galactosylated disaccharides or galactosylated oligosaccharides.

[0067] UDP-galactose can be provided by naturally occurring metabolism in microbial cells, namely through phosphoglucomutase, which catalyzes the interconversion of glucose-1-phosphate and glucose-6-phosphate; UTP-glucose-1-phosphate-uridineyltransferase, which catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP; and UDP-glucose-4-epimerase, which catalyzes the reversible conversion of UDP-glucose to UDP-galactose.

[0068] Intracellular supply of UDP-galactose can be improved through genetic engineering by overexpressing one or more genes encoding phosphoglucomutase, UDP-glucose-1-phosphate-uridine transferase, UDP-glucose-4-epimerase, and their functional variants, in microbial cells, and / or expressing one or more additional copies of one or more genes encoding phosphoglucomutase, UDP-glucose-1-phosphate-uridine transferase, UDP-glucose-4-epimerase, and their functional variants. Examples of genes encoding phosphoglucomutase are the pgm gene (accession number NP_415214) found in *E. coli* K-12, the *E. coli* galU gene (accession number NP_415752) found in *E. coli* K-12, and the *E. coli* galE gene (NP_415280) found in *E. coli* K-12.

[0069] As used herein, the terms “overexpression” or “overexpressing” refer to an enzyme or peptide expressed at a level higher than that measured in wild-type progenitor cells, which are cells of the same species as genetically engineered microbial cells that have not been genetically engineered.

[0070] Additionally and / or alternatively, the supply of intracellular UDP-galactose can be achieved or improved by supplying galactose to the microbial cells via the culture medium in which the microbial cells are cultured. The exogenously supplied galactose is taken up by the microbial cells, then phosphorylated to galactose-1-phosphate, and then converted to UDP-galactose. In this GDP-galactose biosynthesis pathway, the genes encoding the enzymes with the required enzymatic activities are known in the literature (Groissoird et al., "Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway (2003) J. Bacteriol. 185(3) 870-878). The galP gene (accession number NP_417418) encodes a galactose-proton symporter. The galM gene (accession number NP_415277) encodes aldolase 1-epomerase, the galK gene (accession number NP_415278) encodes galactokinase, the galT gene (accession number NP_415279) encodes galactose-1-phosphate uridine transferase, and the galE gene encodes UDP-galactose-4-epomerase.

[0071] Therefore, UDP-galactose biosynthesis can also be provided by expressing genes encoding galactose-proton cotransporters, galactokinase, and galactose-1-phosphate uridine transferase in microbial cells, or by overexpressing genes encoding galactose-proton cotransporters, galactokinase, and galactose-1-phosphate uridine transferase in microbial cells.

[0072] In another embodiment, the genetically engineered microbial cells possess a GDP-fucose biosynthetic pathway for the intracellular formation of GDP-L-fucose (GDP-Fuc). GDP-Fuc is a substrate of fucosyltransferases, the enzymatic activity of which can lead to the formation of fucosylated disaccharides or fucosylated oligosaccharides.

[0073] In an additional and / or alternative embodiment, the genetically engineered microbial cells have a GDP-L-fucose biosynthesis pathway comprising mannose-6-phosphate isomerase, phospmannose mutase, mannose-1-phosphate-guanylate transferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase.

[0074] Intracellular supply of GDP-L-fucose can be improved through genetic engineering, namely by overexpressing one or more genes encoding mannose-6-phosphate isomerase, mannose phosphate mutase, mannose-1-phosphate-guanylate transferase, GDP-mannose-4,6-dehydratase, or GDP-L-fucose synthase in microbial cells, and / or by expressing additional copies of one or more genes encoding mannose-6-phosphate isomerase, mannose phosphate mutase, mannose-1-phosphate-guanylate transferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase or their functional variants in microbial cells. Examples of genes encoding mannose-6-phosphate isomerase are the *E. coli* manA gene (accession number NP_416130) found in *E. coli* K-12, examples of genes encoding mannose phosphate isomerase are the *E. coli* manB gene (accession number NP_416552) found in *E. coli* K-12, examples of genes encoding mannose-1-phosphate-guanylate transferase are the *E. coli* manC gene (accession number NP_416553) found in *E. coli* K-12, examples of genes encoding GDP-mannose-4,6-dehydratase are the *E. coli* gmd gene (accession number NP_416557) found in *E. coli* K-12, and examples of genes encoding GDP-L-fucose synthase are the *E. coli* wcaG gene (accession number NP_416556) found in *E. coli* K-12.

[0075] Additionally and / or alternatively, the supply of GDP-L-fucose can be achieved or improved by supplying L-fucose to the microbial cells via a culture medium in which the microbial cells are cultured. The exogenously supplied L-fucose is taken up by the microbial cells and is first phosphorylated to fucose-1-phosphate by the enzyme fucokinase. Subsequently, fucose-1-phosphate is converted to GDP-L-fucose by the enzymatic activity of the enzyme fucose-1-phosphate guanylate transferase. Genes encoding enzymes with the desired enzymatic activity are known to those skilled in the art. Exemplarily, the fkp gene (accession number WP_010993080), which encodes a bifunctional L-fucokinase / L-fucose-1-phosphate guanylate transferase of Bacteroides fragilis, can be overexpressed in genetically engineered host cells.

[0076] In another embodiment, the genetically engineered microbial cells have a UDP-N-acetylglucosamine biosynthetic pathway for the formation of intracellular UDP-N-acetylglucosamine (UDP-GIcNAc), which is required by N-acetylglucosamine transferase reactions to lead to the formation of, for example, N-acetylglucosamine-modified disaccharides or oligosaccharides.

[0077] In an additional and / or alternative embodiment, the genetically engineered microbial cells have a UDP-N-acetylglucosamine biosynthetic pathway, such as including L-glutamine:D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, and N-acetylglucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase.

[0078] Intracellular supply of UDP-N-acetylglucosamine can be improved by genetic modification, such as by expressing or overexpressing one or more genes or variants of peptides that exhibit the following enzymatic activities: L-glutamine:D-fructose-6-phosphate aminotransferase activity (e.g., E. coli K-12glmS gene (accession number NP_418185)), phosphoglucosamine mutase activity (e.g., E. coli K-12glmM gene (accession number NP_417643)), and N-acetylglucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase (e.g., E. coli K-12glmU gene (accession number NP_418186)).

[0079] In another embodiment, the genetically engineered microbial cells have a CMP-N-acetylneuraminic acid / CMP-sialic acid biosynthetic pathway for the intracellular formation of CMP-N-acetylneuraminic acid (CMP-Neu5Ac), which is required by sialyltransferase reactions to lead to the formation of, for example, sialylated disaccharides or oligosaccharides.

[0080] In an additional and / or alternative embodiment, the genetically engineered microbial cells possess a CMP-N-acetylneuraminic acid biosynthetic pathway, including, for example, L-glutamine:D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate uridineyltransferase / glucosamine-1-phosphate acetyltransferase, UDP-N-acetylglucosamine-2-epomerase, glucosamine-6-phosphate acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase (preferably HAD-like phosphatase), N-acetylglucosamine-2-epomerase, sialic acid synthase, and CMP-sialic acid synthase.

[0081] Intracellular supply of CMP-N-acetylneuraminic acid can be improved through genetic modification, such as expressing or overexpressing one or more genes or variants thereof encoding polypeptides exhibiting the following enzymatic activities: L-glutamine:D-fructose-6-phosphate aminotransferase (e.g., *E. coli* K-12glmS gene), phosphoglucosamine mutase activity (e.g., *E. coli* K-12glmM gene), N-acetylglucosamine-1-phosphate uridineyltransferase / glucosamine-1-phosphate acetyltransferase (e.g., *E. coli* K-12glmU gene), UDP-N-acetylglucosamine-2-epomerase (e.g., *Campylobacter jejuni* neuC gene (accession number AF305571)), glucosamine-6-phosphate acetyltransferase (e.g., *Saccharomyces cerevisiae*). The following genes are used to detect N-acetylglucosamine-6-phosphate phosphatase (e.g., Escherichia coli K-12yihX gene (accession number NP_418321)), N-acetylglucosamine 2-epimerase (e.g., Synechocystis sp. PCC6803 slr1975 gene (accession number BAL35720)), sialic acid synthase (e.g., Campylobacter jejuni neuB gene (accession number AF305571)), and CMP-sialic acid synthase (e.g., Campylobacter jejuni neuA gene (accession number AF305571)).

[0082] Genetically engineered microbial cells may also contain glycosyltransferases. In a preferred embodiment, at least one glycosyltransferase is a fucosyltransferase, sialyltransferase, glucosamine transferase, or galactosyltransferase; more preferably, said at least one glycosyltransferase exhibits β-1,3-galactosyltransferase activity, β-1,4-galactosyltransferase activity, β-1,6-galactosyltransferase activity, β-1,3-N-acetylglucosamine transferase activity, α-2,3-sialyltransferase activity, α-2,6-sialyltransferase activity, α-1,2-fucosyltransferase activity, α-1,3-fucosyltransferase activity, or α-1,4-fucosyltransferase activity. Suitable glycosyltransferases are known to those skilled in the art or can be found in the literature.

[0083] Generally, throughout this disclosure, the term "glycosyltransferase activity" or "glycosyltransferase" means and encompasses enzymes responsible for the biosynthesis of disaccharides, oligosaccharides, and polysaccharides, and that catalyze the transfer of monosaccharide moieties from activated nucleotide monosaccharides / sugars (e.g., UDP-Glc, UDP-Gal, GDP-Fuc, UDP-GlcNAc, CMP-Neu5Ac) to glycosyl acceptor molecules (e.g., monosaccharides, disaccharides, or oligosaccharides).

[0084] In an additional and / or alternative embodiment, the genetically engineered microbial cells synthesize more phosphoenolpyruvate (PEP) than wild-type cells. In another additional and / or alternative embodiment, the genetically engineered microbial cells have been genetically engineered to have an enhanced PEP biosynthetic pathway. For example, the genetically engineered microbial cells have been genetically engineered to have increased phosphoenolpyruvate carboxylkinase activity, for example, by overexpressing the *E. coli* K-12pckA gene (accession number NP_417862) or a functional variant thereof. Preferably, the genetically engineered host cells have been genetically engineered to have increased phosphoenolpyruvate synthase activity, for example because the *E. coli* K-12ppsA gene (accession number NP_416217) encoding phosphoenolpyruvate synthase is overexpressed and / or the non-naturally occurring microorganism contains at least one additional copy of a nucleotide sequence that enables phosphoenolpyruvate synthase expression or a functional variant thereof. Overexpression of pckA or ppsA enhances intracellular PEP synthesis, thereby obtaining more PEP, for example, for the production of sialic acid.

[0085] In an additional and / or alternative implementation, intracellular PEP availability in genetically engineered host cells can be improved by reducing and / or decreasing the activity of phosphoenolpyruvate:glucose phosphotransferase-dependent input proteins, which is required to transfer specific sugars (e.g., glucose) used as carbon and energy sources from the culture medium into the cells. Therefore, to maintain the ability of genetically engineered host cells to use said certain sugars as carbon and energy sources, it is necessary to express or overexpress phosphoenolpyruvate:glucose phosphotransferase-independent input proteins. In an additional and / or alternative implementation, it is necessary to reduce or decrease the expression or overexpression of kinases capable of phosphorylating said certain sugars to achieve the metabolism of said sugars or their accumulation in the cells in their unphosphorylated form.

[0086] Phosphoenolpyruvate: A glucose phosphotransferase-dependent input protein or a component thereof—whose expression and / or activity may be reduced and / or decreased in genetically engineered microbial cells (e.g., Escherichia coli K-12)—contains at least one of the following: glucose PEP-PTS genes ptsG (accession number NP_415619), malX (accession number NP_416138), crr (accession number NP_416912), bglF (accession number NP_418178) and / or fruose PEP-PTS genes fruA (accession number NP_416672), fruB (accession number NP_416674) and / or mannose PEP-PTS genes manX (accession number NP_416331), many (accession number NP_416332), manZ (accession number NP_416333) and / or N-acetylglucosamine PEP-PTS gene nagE (accession number NP_415205).

[0087] Technicians know suitable non-PEP-PTS transporters or variants thereof capable of transferring monosaccharides (glucose and / or fructose and / or galactose and / or N-acetylglucosamine and / or N-acetylneuraminic acid and / or fucose) into microbial cells. Non-limiting examples of genes encoding the aforementioned non-PEP-PTS transporter proteins are: galP of *Escherichia coli* K-12 (SEQ ID NO. 1), glf of *Zymomonas mobilis* (SEQ ID NO. 2), cscB of *Escherichia coli* W (SEQ ID NO. 3), fupL of *Leuconostoc pseudomesenteroides* (SEQ ID NO. 4), lacY of *Escherichia coli* K-12 (SEQ ID NO. 5), fucP of *Escherichia coli* K-12 (SEQ ID NO. 6), nanT of *Escherichia coli* K-12 (SEQ ID NO. 7), nagP of *Xanthomonas campestris* (SEQ ID NO. 8), glcP of *Bifidobacterium longum* NCC2705 (SEQ ID NO. 9), glcP of *Bacillus subtilis* (SEQ ID NO. 10), and *Vibrio parahaemolyticus*. The gene described includes *parahaemolyticus* sglS (SEQ ID NO. 11), *Escherichia coli* K-12 xylE (SEQ ID NO. 12), and *Bacillus subtilis* 168 araE (SEQ ID NO. 13). Therefore, in an additional and / or alternative embodiment, the genetically engineered host cell contains and expresses at least one gene that includes the protein-coding region of the aforementioned gene or a functional variant thereof.

[0088] In a preferred embodiment, suitable non-PEP-PTS-glucose transporters are sugar-promoting diffusion proteins and / or glucose transport permeases. Suitable glucose-promoting fusion proteins are encoded by the *glf* gene of *Fermentomonas motile*. Suitable glucose transport permeases are encoded by the *Escherichia coli* K-12galP gene. Glucose transport permeases are also known as galactose-proton cotransporters or galactose permeases, but they also transport glucose across the cell membrane.

[0089] In another preferred embodiment, suitable non-PEP-PTS-fructose transporters are sugar-promoting diffusion proteins and / or fructose carrier proteins. Suitable fructose-promoting fusion proteins are encoded by the *glf* gene of *Morhizium motiformis*. Suitable fructose carrier proteins are encoded by the *fupL* gene of *Leuconostoc pseudoenteroides*.

[0090] Generally, throughout this disclosure, the terms "increased intracellular availability of phosphate sugars" or "increased / enhanced supply of phosphate sugars" refer to the enhanced ability of genetically modified microbial cells to produce an increased intracellular amount of said phosphate sugars compared to unmodified microbial cells, resulting in increased carbon flux through the biosynthetic pathway of desired carbohydrates. Therefore, the increase in said desired carbohydrates produced by said genetically modified microbial cells is a direct measure of the increase in the intracellular amount of said phosphate sugars. Furthermore, methods for targeting the quantification of intracellular metabolites and / or cellular carbon flux can be found in literature known to those skilled in the art (e.g., and Weckwerth, "Metabolomics Practice: Successful Strategies to Generate and Analyze Metabolic Data", Wiley-VCH, Weinheim, Germany (2013).

[0091] In an additional and / or alternative embodiment, the expression and / or activity of genes and / or proteins that compete with the biosynthesis of desired carbohydrates may be reduced and / or decreased within genetically engineered host cells. Non-limiting examples of such counteraction of multiple protein / one protein activity include at least one selected from: β-galactosidase (e.g., *E. coli* K-12 LacZ (accession number NP_414878)), UDP-glucose:undecylphosphoglucose-1-phosphotransferase (e.g., *E. coli* K-12 WcaJ (accession number NP_416551)), L-fucose isomerase (e.g., *E. coli* K-12 Fucl), fucokinase (e.g., *E. coli* K-12 FucK (accession number NP_417282)), N-acetylglucosamine-6-phosphate deacetylase (e.g., *E. coli* K-12 NagA (accession number NP_417282)), and N-acetylglucosamine-6-phosphate deacetylase (e.g., *E. coli* K-12 NagA (accession number NP_417282)). Accession number NP_415203), glucosamine-6-phosphate deaminase (e.g., Escherichia coli K-12NagB (accession number NP_415204)), N-acetylmnosamine kinase (e.g., Escherichia coli K-12NanK (accession number NP_417689)), N-acetylmnosamine-6-phosphate epimerase (e.g., Escherichia coli K-12NanE (accession number NP_417690)), N-acetylneuraminic acid aldolase (e.g., Escherichia coli K-12NanA (accession number NP_417692)), and sialic acid permease (e.g., Escherichia coli K-12NanT (accession number NP_417691)).

[0092] In an additional and / or alternative implementation, the genetically engineered microbial cells are capable of metabolizing sucrose and therefore contain one or more genes encoding the following:

[0093] (i) a heterologous PTS-dependent sucrose utilization transport system (e.g., the Klebsiella pneumoniae scrYAB gene (SEQ ID NO. 14) or the Salmonella typhimurium scrYAB gene (SEQ ID NO. 15)) composed of sucrose porin (scrY), PTS sucrose-specific transporter subunit II (scrA), and sucrose-6-phosphate hydrolase (scrB), and / or (ii) a heterologous PTS-dependent sucrose transport system (e.g., the Klebsiella pneumoniae scrYA gene or the Salmonella typhimurium scrYA gene) combined with a sucrose phosphate synthase gene (e.g., the Anabaena sp. PCC7120 spsA gene (accession number AJ302071)), and / or (iii) a heterologous PTS-independent sucrose utilization system (e.g., the Escherichia coli W cscBKA gene (SEQ ID NO. 15)). NO.16)) consists of fructose kinase (cscK), sucrose hydrolase (cscA) and sucrose permease (cscB), and / or (iv) a heterologous sucrose utilization system consisting of a sucrose permease gene (e.g., Escherichia coli W cscB) combined with a sucrose phosphorylase gene (e.g., Bifidobacterium adolescentis basP gene (accession number WP_011742626)) or a sucrose synthase gene (e.g., Anabaena susA gene (accession number CAA09297)).

[0094] In an additional and / or alternative embodiment, the microbial cell contains an export protein or permease that exports the target carbohydrate from the cell, preferably a sugar efflux transporter.

[0095] In an additional and / or alternative embodiment, the host cell is a microbial cell, preferably selected from the following genera: *Escherichia*, *Lactobacillus*, *Corynebacterium*, *Bacillus*, *Streptococcus*, *Enterococcus*, *Lactococcus*, and *Clostidium*, and preferably selected from the following species: *Escherichia coli*, *Corynebacterium glutamicum*, *Clotridium cellulolyticum*, *Clotridium ljungdahlii*, *Clotridium autoethanogenum*, *Clotridium acetobutylicum*, *Bacillus subtilis*, *Bacillus megaterium*, and *Lactobacillus casei*. The bacteria include *Lactobacillus casei*, *Lactobacillus acidophilus*, *Lactobacillus helveticus*, *Lactobacillus delbrueckii*, and *Lactococcus lactis*. In another embodiment, the microbial cells are *Escherichia coli*. Other bacterial strains will be recognized by those skilled in the art upon reading this disclosure.

[0096] According to a second aspect, the present invention provides the use of genetically engineered host cells, as described herein, for the production of desired carbohydrates, wherein the host cells are cultured in the presence of a monosaccharide mixture consisting of glucose and at least one second monosaccharide of fructose and galactose. The cells are genetically engineered to exhibit increased intracellular availability of at least one phosphate sugar associated with the production of desired carbohydrates. The phosphate sugar may be selected from phosphoenolpyruvate (PEP), dihydroxyacetone phosphate, fructose-6-phosphate, fructose-1-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, glucose-1-phosphate, galactose-1-phosphate, mannose-1-phosphate, mannose-6-phosphate, glucosamine-6-phosphate, glucosamine-1-phosphate, N-acetylglucosamine-6-phosphate, N-acetylglucosamine-1-phosphate, N-acetylglucosamine-6-phosphate, UDP-glucose, UDP-galactose, CMP-N-acetylneuraminic acid, GDP-mannose, GDP-fucose, and UDP-N-acetylglucosamine.

[0097] According to a third aspect, the present invention provides a method for producing desired carbohydrates through fermentation, comprising the following steps:

[0098] a) Provide genetically engineered microorganisms capable of producing desired carbohydrates, wherein the microorganisms exhibit increased intracellular availability of at least one phosphate sugar due to reduced and / or decreased expression and / or activity of proteins that consume intracellular phosphate sugars within the cells of the engineered microorganisms.

[0099] b) Cultivate the genetically engineered microorganism in a culture medium that allows the production of the desired carbohydrates, wherein the primary carbon source is a mixture of monosaccharides consisting of glucose and at least one secondary monosaccharide of fructose and galactose;

[0100] c) Recover the desired sugar from the culture medium.

[0101] In an additional and / or alternative embodiment, the target carbohydrate is a human lactose oligosaccharide or a building block thereof. Table 1 discloses a list of target carbohydrates that can be produced using genetically modified microbial cells and / or methods described herein. Preferably, the desired carbohydrate is selected from 2'-fucosylated lactose, 3-fucosylated lactose, 2',3-difucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, 3-fucosylated-3'-sialylated lactose, lact-N-tetrasaccharide, lact-N-neotetrasaccharide, lact-N-fucopentose I, lact-N-fucopentose II, lact-N-fucopentose III, lact-N-fucopentose V, lact-N-difucosylated hexose I, lact-N-difucosylated hexose II, lact-N-sialylated pentose LSTa, LSTb, LSTc.

[0102] Preferably, the mixture of glucose and at least one other monosaccharide is a mixture of glucose and fructose, preferably obtained by hydrolyzing sucrose.

[0103] In an additional and / or alternative embodiment, sucrose hydrolysis is incomplete, resulting in a significant amount of sucrose remaining in the feedstock. Therefore, the sucrose content in the hydrolyzed and / or heat-sterilized feedstock is greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or greater than 80%.

[0104] In an additional and / or alternative implementation, microbial cells are cultured without an exogenous supply of receptor substrates, such as N-acetylglucosamine or lactose, particularly when the cultured microbial cells are intended to produce the desired oligosaccharide.

[0105] The invention will be described with reference to specific embodiments and accompanying drawings, but is not limited thereto; rather, it is defined solely by the claims. Furthermore, the terms "first," "second," etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe an order in time, space, sequence, or any other manner. It should be understood that the terms thus used are interchangeable where appropriate, and embodiments of the invention described herein can operate in orders other than those described or illustrated herein.

[0106] It should be noted that the term "comprising" as used in the claims should not be construed as limited to the manner listed thereafter; it does not exclude other elements or steps. Therefore, it is interpreted as indicating the presence of the stated feature, integer, step, or component, but does not exclude the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the expression "device comprising means A and B" should not be limited to devices consisting solely of components A and B. This means that, for the purposes of this invention, the only relevant components of the device are A and B.

[0107] Throughout this specification, references to "one embodiment" or "an embodiment" mean that at least one embodiment of the invention includes the specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "one embodiment" or "an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, specific features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art based on this disclosure.

[0108] Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, its figures, or description in order to simplify the disclosure and aid in understanding one or more aspects of the invention. However, this method of disclosure should not be construed as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the following claims, aspects of the invention lie in fewer than all features of a single foregoing disclosed embodiment. Therefore, the claims following the detailed description are expressly incorporated into the detailed description, each claim in itself being a separate embodiment of the invention.

[0109] Furthermore, while some embodiments described herein include some features included in other embodiments but not others, combinations of features from different embodiments are within the scope of this invention and form different embodiments, as will be understood by those skilled in the art. For example, in the following claims, any claimed embodiment may be used in any combination.

[0110] Furthermore, this document describes some embodiments as methods or combinations of method elements that can be implemented by a processor of a computer system or by other means of performing the function. Therefore, a processor having the necessary instructions to perform such methods or method elements constitutes a means for performing said methods or method elements. Moreover, the elements of the apparatus embodiments described herein are examples of means for implementing the invention and performing the functions performed by said elements.

[0111] Numerous specific details are set forth in the specification and accompanying drawings provided herein. However, it should be understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail to avoid obscuring the understanding of this specification.

[0112]

[0113]

[0114]

[0115]

[0116] Table 1: A non-limiting list of target carbohydrates that can be produced by using genetically modified microbial cells and / or methods described herein.

[0117] The invention will now be described by way of specific description of several embodiments thereof. It will be apparent that other embodiments of the invention may be devised based on the knowledge of those skilled in the art without departing from the true spirit of the invention or its technical teachings, and the invention is limited only by the terminology of the appended claims.

[0118] In one implementation, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source to treat individuals with the genotype nagABE. - manXYZ -The *E. coli* strain is metabolically engineered into an efficient producer of N-acetylglucosamine (GlcNAc). Therefore, heterologous expression of the gene encoding glucosamine-6-phosphate acetyltransferase is necessary, which is capable of transferring acetate from acetyl-CoA to glucosamine-6-phosphate, thereby producing N-acetylglucosamine-6-phosphate. In a preferred embodiment, the producing strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf. This further genetic modification allows the engineered producing strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic interference with the strain but increasing the precursor supply (fructose-6-phosphate) for N-acetylglucosamine production. In an additional implementation, a gene encoding glutamine-fructose-6-phosphate aminotransferase (e.g., Escherichia coli GrmS) and / or a gene encoding an HAD-like sugar phosphatase (e.g., Escherichia coli YihX, Escherichia coli YqaB) capable of dephosphorylating N-acetylglucosamine-6-phosphate to N-acetylglucosamine are expressed / overexpressed to promote GlcNAc synthesis.

[0119] In another embodiment, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and lactose as the acceptor substrate, to target individuals with the lacY genotype. + lacZ - fuclK - wcaJ - The *E. coli* strain is metabolically engineered into an efficient producer of L-fucose. Therefore, overexpression of at least one of the *E. coli* genes manA, manC, manB, gmd, and wcaG, as well as expression of heterologous α-1,2-fucosyltransferase (capable of transferring fucose from GDP-fucose to lactose, thereby producing 2'-fucosyllactose) and α-1,2-fucosidase (capable of releasing free L-fucose from 2'-fucosyllactose), is necessary. In a preferred embodiment, the producing strain is further engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf. This further genetic modification allows the engineered producing strain to be cultured on a mixed monosaccharide feedstock (such as hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (fructose-6-phosphate) for L-fucose production.

[0120] In another implementation, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, to produce nanKETA genotypes. - ,nagAB -The *E. coli* strain is metabolically engineered to be an efficient producer of N-acetylneuraminic acid (Neu5Ac). Therefore, overexpression of at least one gene encoding (i) glucosamine-6-phosphate acetyltransferase and N-acetylglucosamine-2-epomerase and N-acetylneuraminic acid synthase, or (ii) UDP-N-acetylglucosamine-2-epomerase and N-acetylneuraminic acid synthase, is necessary. Since N-acetylneuraminic acid synthesis is a phosphoenolpyruvate (PEP)-dependent process, competitive reactions leading to PEP depletion are preferably avoided. Therefore, in a preferred embodiment, the producing strain is further engineered via a phosphoenolpyruvate:glucose phosphotransferase-dependent mechanism by reducing and / or decreasing the input of the carbon and energy sources, for example, by reducing and / or decreasing the expression of the glucose PEP permease gene ptsG and / or the fructose PEP permease gene fruA and / or the mannose PEP permease gene manXYZ. With the expression / overexpression of at least one gene encoding a phosphoenolpyruvate:glycophosphotransferase-independent (non-PEP-PTS) transporter (enabling the transfer of monosaccharides to engineered cells), as well as fructose kinases (such as the *E. coli* W cscK gene) and glucokinases (such as the *E. coli* K-12glk gene) (capable of activating fructose to fructose-6-phosphate and glucose to glucose-6-phosphate, respectively), this further genetic modification allows engineered production strains to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the supply of precursors (fructose-6-phosphate, phosphoenolpyruvate) for N-acetylneuraminic acid production.

[0121] In another implementation, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and GlcNAc is used as the acceptor substrate to target individuals with the genotype nagABE. - manXYZ - lacZ -Escherichia coli strains are metabolically engineered into efficient producers of N-acetyllactosamine (LacNAc). Therefore, expression / overexpression of genes encoding glucosamine-6-phosphate acetyltransferase, a phosphoenolpyruvate:glucose phosphotransferase-independent transporter (capable of transferring N-acetylglucosamine into cells), and β-1,4-galactosyltransferase (capable of transferring galactose from UDP-Gal to free N-acetylglucosamine, thereby producing N-acetyllactosamine) is necessary. In a preferred embodiment, the producing strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate isomerase gene pgi and / or the glucose-6-phosphate dehydrogenase gene zwf. This further genetic modification allows the engineered producing strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (glucose-6-phosphate) for N-acetyllactosamine production. In an additional implementation, at least one of the E. coli genes pgm, galU, and galE is overexpressed to promote the synthesis of UDP-Gal.

[0122] In another embodiment, the nagAB genotype is expressed through total fermentation using a mixture of monosaccharide feedstocks (e.g., hydrolyzed sucrose) as the primary carbon and energy source. - The *E. coli* strain is metabolically engineered to be an efficient producer of lacto-N-biose (LNB). Therefore, it is necessary to express / overexpress genes encoding glucosamine-6-phosphate acetyltransferase (capable of transferring acetate from acetyl-CoA to glucosamine-6-phosphate, thereby producing N-acetylglucosamine-6-phosphate), HAD-like glucose phosphatase (capable of dephosphorylating N-acetylglucosamine-6-phosphate, thereby producing N-acetylglucosamine), and β-1,3-galactosyltransferase (capable of transferring galactose from UDP-Gal to free N-acetylglucosamine, thereby producing LNB). In a preferred embodiment, the producing strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf. This further genetic modification allows engineered production strains to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the supply of precursors (fructose-6-phosphate and glucose-6-phosphate) for lacto-N-biose production. In an additional embodiment, at least one of the *E. coli* genes glmS, pgm, galU, and galE is overexpressed to promote the synthesis of GlcNAc and / or UDP-Gal.

[0123] In another embodiment, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and lactose as the acceptor substrate, to target individuals with the lacY genotype. + lacZ - nanKETA - ,nagAB - Escherichia coli strains were metabolically engineered into efficient producers of 3'-sialyl lactose. Therefore, the N-acetylneuraminic acid (3'SL) producing strain was genetically engineered by expressing heterologous CMP-N-acetylneuraminic acid synthase, N-acetylneuraminic acid synthase, and α-2,3-sialyl transferase (capable of transferring N-acetylneuraminic acid from CMP-Neu5Ac to lactose, thereby producing 3'-sialyl lactose). Regarding the synthesis of N-acetylneuraminic acid, the production of 3'SL is a phosphoenolpyruvate (PEP)-dependent method. Therefore, in a preferred embodiment, the 3'SL producing strain is further engineered via a phosphoenolpyruvate:glucose phosphotransferase-dependent mechanism by reducing and / or decreasing the input of the carbon and energy sources, for example, by reducing and / or decreasing the expression of the glucose PEP permease gene ptsG and / or the fructose PEP permease gene fruA and / or the mannose PEP permease gene manXYZ. With the expression / overexpression of at least one gene encoding a phosphoenolpyruvate:glycophosphotransferase-independent (non-PEP-PTS) transporter (enabling the transfer of monosaccharides to engineered cells) and a fructose kinase (e.g., the *E. coli* W cscK gene) and a glucokinase (e.g., the *E. coli* K-12glk gene) (capable of activating fructose to fructose-6-phosphate and glucose to glucose-6-phosphate, respectively), this further genetic modification allows engineered production strains to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (fructose-6-phosphate, phosphoenolpyruvate) for 3'-sialic acid lactose production.

[0124] In another embodiment, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and lactose as the acceptor substrate, to target individuals with the lacY genotype. + lacZ - fuclK - wcaJ -Escherichia coli strains are metabolically engineered to be efficient producers of 3-fucosylated lactose. Therefore, overexpression of at least one of the E. coli genes manA, manC, manB, gmd, and wcaG, as well as expression of a heterologous α-1,3-fucosyltransferase (capable of transferring fucose from GDP-fucoose to lactose, thereby producing 3-fucosylated lactose) is necessary. In a preferred embodiment, the producing strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf. This further genetic modification allows the engineered producing strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (fructose-6-phosphate) for 3-fucosylated lactose production.

[0125] In one implementation, a mixture of monosaccharide feedstocks (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and lactose as the acceptor substrate, to target individuals with the lacY genotype. + lacZ - nagB - wcaJ - The *E. coli* strain is metabolically engineered to efficiently produce lactotrisaccharide II (LNT-II). Therefore, expression of heterologous β-1,3-N-acetylglucosamine transferase (capable of transferring N-acetylglucosamine from UDP-GlcNAc to lactose, thereby generating LNT-II) is necessary. In a preferred embodiment, the production strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase gene pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf. This further genetic modification allows the engineered production strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (fructose-6-phosphate) for LNT-II production. In an additional embodiment, overexpression of at least one of the *E. coli* genes glmS, glmU, and glmM promotes UDP-GlcNAc synthesis.

[0126] In another embodiment, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and lactose as the acceptor substrate, to target individuals with the lacY genotype. + lacZ - nagB - wcaJ -Escherichia coli strains are metabolically engineered to be efficient producers of lacto-N-tetrasaccharides (LNTs). Therefore, it is necessary to express heterologous β-1,3-N-acetylglucosamine transferase (capable of transferring N-acetylglucosamine from UDP-GlcNAc to lactose) and β-1,3-galactosyltransferase (capable of transferring galactose from UDP-galactose to lacto-N-trisaccharide II, thereby generating lacto-N-tetrasaccharides). In a preferred embodiment, the producing strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf and / or the glucose-6-phosphate isomerase gene pgi. This further genetic modification allows the engineered producing strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (fructose-6-phosphate and glucose-6-phosphate) for lacto-N-tetrasaccharide production. In an additional embodiment, at least one of the E. coli genes glmS, glmU, glmM, pgm, galU, and galE is overexpressed to promote the synthesis of UDP-GlcNAc and / or UDP-Gal.

[0127] In another embodiment, the lacY genotype is expressed through total fermentation using a mixture of monosaccharide feedstocks (e.g., hydrolyzed sucrose) as the primary carbon and energy source. + lacZ - nagB - wcaJ -A strain of *E. coli* was metabolically engineered to efficiently produce lact-N-neotetrasaccharide (LNnT). Therefore, this LNnT-producing strain was genetically engineered via a phosphoenolpyruvate:sugar phosphotransferase-dependent mechanism by reducing and / or decreasing glucose input, for example by reducing and / or decreasing the expression of the glucose PEP permease gene ptsG, while simultaneously expressing at least one gene encoding a phosphoenolpyruvate:sugar phosphotransferase-independent (non-PEP-PTS) transporter, enabling glucose transfer to the engineered cells. Furthermore, the expression of the glucokinase gene glk and / or the glucose dehydrogenase gene gcd was reduced and / or eliminated. Essentially, heterologous expression of β-1,4-galactosyltransferase (capable of transferring galactose from UDP-galactose to glucose, thereby producing lactose), β-1,3-N-acetylglucosamine transferase (capable of transferring N-acetylglucosamine from UDP-GlcNAc to lactose, thereby producing lact-N-trisaccharide II), and β-1,4-galactosyltransferase (capable of transferring galactose from UDP-galactose to lact-N-trisaccharide II, thereby producing lact-N-neotetrasaccharide) is necessary. In a preferred embodiment, the production strain is further engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf and / or the glucose-6-phosphate isomerase gene pgi. This further genetic modification allows engineered production strains to be cultured on a mixed monosaccharide feedstock (such as hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (glucose and fructose-6-phosphate and glucose-6-phosphate) to produce lacto-N-neotetrasaccharides via total fermentation. In an additional embodiment, overexpression of at least one of the *E. coli* genes glmS, glmU, glmM, pgm, galU, and galE promotes the synthesis of UDP-GlcNAc and / or UDP-Gal.

[0128] In another embodiment, the lacY genotype is expressed through total fermentation using a mixture of monosaccharide feedstocks (e.g., hydrolyzed sucrose) as the primary carbon and energy source. - lacZ - fuclK - wcaJ -Metabolic engineering of *E. coli* strains was performed to efficiently produce 2'-fucosylated lactose. This involved reducing and / or eliminating the expression of the glucokinase gene *glk* and / or the glucose dehydrogenase gene *gcd*, as well as the activity of the phosphoenolpyruvate:glucose phosphotransferase-dependent mechanism. Furthermore, the *E. coli* strain was expressed or overexpressed with a phosphoenolpyruvate:glucose phosphotransferase-independent (non-PEP-PTS) transporter gene. Additionally, at least one of the *E. coli* genes *manA*, *manC*, *manB*, *gmd*, *wcaG*, *pgm*, *galU*, and *galE*, as well as heterologous β-1,4-galactosyltransferase (capable of transferring galactose from UDP-galactose to glucose to produce lactose) and α-1,2-fucosylated enzyme (capable of transferring fucose from GDP-fucoose to lactose to produce 2'-fucolactose). In a preferred embodiment, the production strain is further engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB and / or the glucose-6-phosphate dehydrogenase gene zwf and / or the glucose-6-phosphate isomerase gene pgi. This further genetic modification allows the engineered production strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (glucose and fructose-6-phosphate and glucose-6-phosphate) for the production of 2'-fucosylated lactose via total fermentation.

[0129] In another embodiment, a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) is used as the primary carbon and energy source, and lactose as the acceptor substrate, to target individuals with the lacY genotype. + lacZ - fuclK - wcaJ - manA -Escherichia coli strains were metabolically engineered to be efficient producers of 2',3-difucosylated lactose. Therefore, the expression of genes exhibiting fructose kinase activity (e.g., the E. coli K-12mak gene) and the activity of the phosphoenolpyruvate:fructose phosphotransferase-dependent mechanism were reduced and / or eliminated. This was achieved by expressing / overexpressing at least one phosphoenolpyruvate:fructose phosphotransferase-independent (non-PEP-PTS) transporter (capable of transferring fructose to engineered cells), mannose isomerase (capable of converting fructose to mannose) (e.g., E. coli BL21 yihS, Agrobacterium radiobacter M-1manI), or mannose kinase (e.g., Prevotella bryantii B14 manK, Arthrobacter sp. strain KM). The cell is further genetically engineered with genes for manK (capable of activating mannose to mannose-6-phosphate) and at least one heterofucosyltransferase exhibiting α-1,2-fucosyltransferase and / or α-1,3-fucosyltransferase activity (capable of transferring fucose from GDP-fucoose to lactose and / or 2'-fucosyllactose and / or 3-fucosyllactose, thereby producing 2'3-difucosyllactose). These further genetic modifications allow the engineered production strain to be cultured on a mixed monosaccharide feedstock (e.g., hydrolyzed sucrose) as the primary carbon and energy source, while preventing metabolic disruption of the strain but increasing the precursor supply (mannose / mannose-6-phosphate) for 2'3-difucosyllactose production. In an additional embodiment, at least one of the *E. coli* genes manC, manB, gmd, and wcaG is overexpressed to promote GDP-Fuc synthesis.

[0130] Non-restricted examples of suitable proteins exhibiting mannose isomerase (ManI) or mannose kinase (ManK) activity can be found in the literature (Hirose et al., Biosci Biotechnol Biochem. 2001 Mar; 65(3):658-61.; Hirose et al., Biotechnol Lett. 2003 Feb; 25(4):349-52.; Patel et al., Appl Environ Microbiol. 2011 May; 77(10):3343-50.; Hu et al., Int J Biol Macromol. 2016 Aug; 89:328-35.; Huang et al., Appl Microbiol Biotechnol. 2018 Mar; 102(5):2051-2062.; Mukai et al., Appl Environ Microbiol. 2003Jul;69(7):3849-57.; Fields and Russel, Microbiology.2001Apr;147(Pt 4):1035-43.; Kroschewski et al., Mol Biochem Parasitol. 2000Jan 5;105(1):71-80.).

[0131] In another embodiment, the nagAB genotype is expressed through total fermentation using a mixture of monosaccharide feedstocks (e.g., a mixture of hydrolyzed sucrose and hydrolyzed lactose) as the primary carbon and energy source. - fuclK - wcaJ -A strain of *E. coli* was metabolically engineered into an efficient producer of H antigen type I (HA-TI). Therefore, this producing strain was genetically engineered by reducing and / or decreasing glucose input via a phosphoenolpyruvate:glucose phosphotransferase through a dependent mechanism, while simultaneously expressing / overexpressing at least one gene encoding a non-PEP-PTS transporter and a monosaccharide kinase (enabling the transfer of glucose and / or galactose into engineered cells and activating these monosaccharides to their phosphorylated forms, respectively). Furthermore, the production strain is expressed / overexpressed with at least one gene encoding glucosamine-6-phosphate acetyltransferase (capable of transferring acetate from acetyl-CoA to glucosamine-6-phosphate, thereby producing N-acetylglucosamine-6-phosphate), HAD-like sugar phosphatase (capable of dephosphorylating N-acetylglucosamine-6-phosphate, thereby producing N-acetylglucosamine), hetero-β-1,3-galactosyltransferase (capable of transferring galactose from UDP-galactose to N-acetylglucosamine, thereby producing lacto-N-biose), and α-1,2-fucosyltransferase (capable of transferring fucose from GDP-fucose to lacto-N-biose, thereby producing H antigen type I). In a preferred embodiment, the production strain is further genetically engineered by reducing and / or decreasing the expression of the phosphofructokinase genes pfkA and / or pfkB. These genetic modifications allow engineered production strains to be cultured on a mixture of monosaccharide feedstocks (such as a mixture of hydrolyzed sucrose and hydrolyzed lactose) as the primary carbon and energy source, while preventing metabolic disruption but increasing the supply of precursors (N-acetylglucosamine, fructose-6-phosphate, and galactose-1-phosphate) for H antigen type I production. In an additional embodiment, at least one of the *E. coli* genes glmS, galT, galE, manC, manB, gmd, and wcaG is overexpressed to promote the synthesis of GlcNAc and / or UDP-Gal and / or GDP-Fuc.

[0132] Reference Figure 1 An exemplary natural microbial cell is schematically illustrated. This microbial cell is capable of consuming a mixture of glucose and fructose to generate tiny intracellular pools of glucose-6-phosphate and fructose-6-phosphate. The microbial cell expresses polynucleotides encoding the phosphoenolpyruvate:glucose phosphotransferase system (PTS) for the infusion of glucose and fructose into the cell. The reaction products primarily enter glycolysis.

[0133] Figure 2An exemplary microbial cell of the present invention is schematically illustrated, which, when cultured on a mixed feed consisting of glucose and fructose, is capable of highly supplying glucose-6-phosphate. Because the expression of glucose-6-phosphate isomerase Pgi and / or glucose-6-phosphate dehydrogenase Zwf has been reduced or decreased by deletion, functional inactivation, or silencing of the pgi and / or zwf genes, the microbial cell can utilize any glucose-6-phosphate produced by introducing glucose into the cell to generate UDP-Glc and / or UDP-Gal. In variants of the microbial cell ( Figure 3 In some microbial cell variants, the phosphoenolpyruvate:sugar phosphotransferase system for fructose has been disabled, for example by the deletion of the fruA gene. Instead, fructose enters the cell via a phosphoenolpyruvate:sugar phosphotransferase-independent (non-PEP-PTS) transporter and is activated by fructose kinases (e.g., *E. coli* WCscK) to fructose-6-phosphate. Figure 4 In some cases, the cell's phosphoenolpyruvate:glucose phosphotransferase system for glucose has also been disabled, for example by the deletion of the ptsG gene. Instead, glucose enters the cell via a phosphoenolpyruvate:glucose phosphotransferase-independent (non-PEP-PTS) transporter and is activated to glucose-6-phosphate by glucokinases (such as E. coli K-12Glk).

[0134] Figure 5 An exemplary microbial cell of the present invention is schematically illustrated, which, when cultured on a mixed feed consisting of glucose and fructose, is able to supply glucose-6-phosphate and fructose-6-phosphate to a higher degree. The cell's phosphoenolpyruvate:sugar phosphotransferase system for fructose has been disabled, for example by deletion of the fruA gene. Instead, fructose enters the cell via a phosphoenolpyruvate:sugar phosphotransferase-independent (non-PEP-PTS) transporter and is activated to fructose-6-phosphate by fructose kinases (such as *E. coli* W CscK). In variants of the microbial cell ( Figure 6 ), by deleting, functionally inactivating, or silencing the pfkA and / or pfkB and / or zwf genes, or by reducing or decreasing the expression of phosphofructokinase Pfk and / or glucose-6-phosphate dehydrogenase Zwf, thereby increasing the availability of glucose-6-phosphate and fructose-6-phosphate, which can be utilized by microbial cells to produce UDP-Gal and / or GDP-Fuc and / or UDP-GlcNAc and / or CMP-Neu5Ac. In variants of microbial cells ( Figure 7Cells express polynucleotides encoding the phosphoenolpyruvate:glucose phosphotransferase system (PTS) for the ingestion of glucose and fructose into cells. The availability of glucose-6-phosphate and fructose-6-phosphate has been increased by deleting, functionally inactivating, or silencing the pfkA and / or pfkB and / or zwf genes, thereby reducing or decreasing the expression of phosphofructokinase Pfk and / or glucose-6-phosphate dehydrogenase Zwf.

[0135] Figure 8 An exemplary microbial cell of the present invention is illustrated schematically, which, when cultured on a mixed feed consisting of glucose and fructose, is capable of supplying glucose-6-phosphate and fructose-6-phosphate to a greater extent. The cell expresses polynucleotides encoding the phosphoenolpyruvate:glucose phosphotransferase system (PTS) for the infusion of glucose and fructose into the cell. Expression of phosphofructokinase Pfk and / or glucose-6-phosphate isomerase Pgi and / or glucose-6-phosphate dehydrogenase Zwf has been reduced or decreased by deletion, functional inactivation, or silencing of the pfkA and / or pfkB and / or pgi and / or zwf genes. With overexpression of fructose-1,6-bisphosphatases (such as glpX, fbp), increased availability of glucose-6-phosphate and fructose-6-phosphate is generated and can be utilized by the microbial cell to produce UDP-Gal and / or GDP-Fuc and / or UDP-GlcNAc and / or CMP-Neu5Ac.

[0136] Figure 9 An exemplary microbial cell of the present invention is illustrated schematically. When cultured on a mixed feed consisting of glucose and fructose, the microbial cell is capable of producing high availability of glucose-6-phosphate, fructose-6-phosphate, and phosphoenolpyruvate. The cell's phosphoenolpyruvate:glycophosphotransferase system for glucose and fructose has been disabled, for example by the deletion of the ptsG and fruA genes, respectively. Instead, fructose and glucose enter the cell via phosphoenolpyruvate:glycophosphotransferase-independent (non-PEP-PTS) transporters, and are activated by fructose kinases (such as *E. coli* W CscK) and glucokinases (such as *E. coli* K-12Glk) to form fructose-6-phosphate and glucose-6-phosphate, respectively. Furthermore, PEP consumption of the phosphoenolpyruvate:glycophosphotransferase system is avoided. Increased availability of glucose-6-phosphate and fructose-6-phosphate, as well as phosphoenolpyruvate, is achieved through the reduction and / or decrease of expression of phosphofructokinase Pfk and / or glucose-6-phosphate dehydrogenase Zwf, resulting from the deletion, functional inactivation, or silencing of the pfkA and / or pfkB and / or zwf genes. This can be utilized by microbial cells to produce UDP-Gal and / or GDP-Fuc and / or UDP-GlcNAc and / or CMP-Neu5Ac.

[0137] Figure 10 Another exemplary microbial cell of the present invention is illustrated schematically, which, when cultured on a mixed feed consisting of glucose and fructose, is capable of producing increased availability of free glucose and glucose-6-phosphate and fructose-6-phosphate. The cell's phosphoenolpyruvate:glycophosphotransferase system for glucose and fructose has been disabled, for example by deletion of the ptsG and fruA genes, respectively. Instead, fructose and glucose enter the cell via phosphoenolpyruvate:glycophosphotransferase-independent (non-PEP-PTS) transporters. With the deletion of the glk gene, the microbial cell is genetically engineered to make free glucose monomers available in the cell. Conversely, fructose is activated to fructose-6-phosphate by fructose kinases (such as *E. coli* W CscK). The expression of phosphofructose kinase Pfk and / or glucose-6-phosphate dehydrogenase Zwf is reduced or decreased by deletion, functional inactivation, or silencing of the pfkA and / or pfkB and / or zwf genes. Overall, this resulted in improved intracellular supply of free glucose and glucose-6-phosphate, as well as fructose-6-phosphate. Glucose can serve as a substrate for various glycosylation reactions, while glucose-6-phosphate and fructose-6-phosphate can be utilized by microbial cells to produce UDP-Gal and / or GDP-Fuc and / or UDP-GlcNAc and / or CMP-Neu5Ac. Furthermore, PEP depletion of the phosphoenolpyruvate:glucose phosphotransferase system is avoided.

[0138] Figure 11 Another exemplary microbial cell of the present invention is illustrated schematically, which, when cultured on a mixed feed consisting of glucose and fructose, achieves increased intracellular availability of free mannose and / or mannose-6-phosphate. The cell's phosphoenolpyruvate:glucose phosphotransferase system for fructose has been disabled, for example by deletion of the fruA gene and / or the manXYZ gene. Instead, fructose enters the cell via sugar permeases and / or channels. The microbial cell is genetically engineered to make mannose-6-phosphate available within the cell by deleting genes exhibiting frucokinase activity (e.g., mak) and / or mannose-6-phosphate isomerase activity (e.g., manA) and by expressing / overexpressing suitable mannose isomerases (ManI) and mannose kinases (ManK). This can be utilized by the microbial cell to produce GDP-Man and / or GDP-Fuc.

[0139] Figure 12Another exemplary microbial cell of the present invention is illustrated schematically, which, when cultured on a mixed feed consisting of glucose, fructose, and galactose, is capable of producing higher intracellular availability of free fructose-6-phosphate and / or galactose-1-phosphate. The cell's phosphoenolpyruvate:glycophosphotransferase system for glucose has been disabled, for example by deletion of the ptsG gene. Instead, glucose enters the cell via a phosphoenolpyruvate:glycophosphotransferase-independent (non-PEP-PTS) transporter. Expression of phosphofructokinase Pfk has been reduced or decreased by deletion, functional inactivation, or silencing of the pfkA and / or pfkB genes. With the expression / overexpression of suitable non-PEP-PTS transporters and monosaccharide kinases, enabling the transfer of galactose to engineered cells and its activation to galactose-1-phosphate, the microbial cell has been genetically engineered to make fructose-6-phosphate and galactose-1-phosphate available within the cell. These can be utilized by microbial cells to produce GlcNAc and / or GDP-Fuc and / or UDP-Gal and / or CMP-Neu5Ac.

[0140] According to a fourth aspect, the present invention provides the use of desired carbohydrates in pharmaceutical and / or nutritional compositions, wherein the desired carbohydrates are preferably produced by the method according to the invention or by genetically engineered host cells.

[0141] Example

[0142] Example 1 - Preparation of mixed monosaccharide raw materials

[0143] A 50% (w / v) sucrose solution was prepared by dissolving 500 g of sucrose in water. The final solution volume was 1 L. The pH was adjusted by using 96% (v / v) sulfuric acid at a temperature of 30°C to 35°C. The solution was then sterilized in a vertical autoclave (Systec VX-65, Linden, Germany) at 121°C for 45 min. Samples were taken before and after heat sterilization and kept frozen before high-performance liquid chromatography (HPLC) analysis. HPLC was performed using a RID-10A refractive index detector (Shimadzu, Germany) connected to a Shimadzu HPLC system and a Waters XBridge Amide 3.5 μm (250 x 4.6 mm) column (Eschborn, Germany). Isocratic elution was performed at 35°C using 30% solvent A (50% (v / v) acetonitrile dissolved in double-distilled water, 0.1% (v / v) NH4OH) and 70% solvent B (80% (v / v) acetonitrile dissolved in double-distilled water, 0.1% (v / v) NH4OH) at a flow rate of 1.4 mL / min. -1The sample was removed by solid-phase extraction on an ion-exchange matrix (Strata ABW, Phenomenex). 10 μL of sample (1:5 dilution) was applied to the column. Finally, the relative amounts of sugars detected were determined. As shown in Table 2, prior to heat treatment, the conversion of sucrose to the monosaccharides glucose and fructose increased with decreasing solution pH. When acidified with sulfuric acid, complete sucrose cleavage was observed at pH ≤ 3.50.

[0144]

[0145] Table 2: Relative amounts of sugar detected in pH-adjusted 50% (w / v) sucrose solutions before and after heat sterilization. pH adjustment was performed using 96% (v / v) sulfuric acid. The percentage of sugar detected by HPLC is described (area under the curve; AUC).

[0146] Example 2 - Raw material-dependent growth of various gene-deleted strains

[0147] A comparison was made between wild-type Escherichia coli strain BL21(DE3) and mutant strain Escherichia coli pfkA. - (△pfkA), Escherichia coli pfkB - (△pfkB), Escherichia coli pfkA - pfkB - Growth behavior of (△pfkA△pfkA). Genome deletion was performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). All strains were cultured at 30°C in 100 mL shake flasks containing 20 mL of mineral salt medium at a concentration of 7 g·L⁻¹. - 1 NH4H2PO4, 7 g·L -1 K2HPO4, 2g·L -1 KOH, 0.3 g·L -1 Citric acid, 2 g·L -1 MgSO4×7·H2O and 0.015 g·L -1 CaCl2×6·H2O, add 1 mL·L -1 Trace element solution (54.4 g·L) -1 Ferric ammonium citrate, 9.8 g·L -1 MnCl2×4·H2O, 1.6 g·L -1 CoCl2×6·H2O, 1g·L -1 CuCl2×2·H2O, 1.9 g·L -1 H3BO3, 9g·L -1ZnSO4×7·H2O, 1.1 g·L -1 Na₂MoO₄×2·H₂O, 1.5 g·L -1 Na2SeO3, 1.5 g·L -1 NiSO4×6·H2O was prepared, containing 2% (m / v) glucose (A) or 1% (w / v) glucose / 1% (w / v) fructose (B) as a carbon source. The culture was inoculated to an OD of 0.1 and measured by OD... 600 Growth and development were measured and monitored for more than 26 hours. For example... Figure 2 As shown, when glucose is provided as the sole carbon and energy source, *E. coli* pfkA... - pfkB - It showed almost no growth, and when mixed monosaccharide feedstock was available, its growth was indistinguishable from that of wild-type strains and single deletion mutants.

[0148] Example 3 - Production of 2'-fucosylated lactose by engineered Escherichia coli strains

[0149] By overexpressing enzymes for the de novo synthesis of GDP-fucose (ManB, ManC, Gmd, WcaG), the 2'-fucosyltransferase gene wbgL from *E. coli* O126, the sugar efflux transporter yberc0001_9420 from *Yersinia bercovieri* ATCC 43970, and the csc gene cluster from *E. coli* W (accession number CP002185.1) (containing genes for sucrose permease, fructose kinase, sucrose hydrolase, and transcriptional repressors (genes cscB, cscK, cscA, and cscR, respectively), further genetic engineering revealed the genotype lacY. + lacZ - fuclK - wcaJ - pfkA -The *E. coli* BL21(DE3) strain was used to enable the strain to grow on sucrose as the sole carbon source. Genome deletion was performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). Genome integration of heterologous genes was performed via transposition. Linear DNA fragments were integrated using the EZ-Tn5™ transposase (Epicentre, USA) or a highly active C9- mutant of the sailor transposase Himar1 (Proc. Natl. Acad. Sci. 1999, USA 96:11428-11433) was used for transposition. These genes were codon-optimized for expression in *E. coli* and synthesized by GenScript. All obtained strains were cultured at 30°C in 100 mL shake flasks containing 20 mL of mineral salt medium. The medium contained 3 g / L KH₂PO₄, 12 g / L K₂HPO₄, 5 g / L (NH₄)₂SO₄, 0.3 g / L citric acid, 2 g / L MgSO₄×7H₂O, 0.1 g / L NaCl, and 0.015 g / L CaCl₂×6H₂O, and 1 mL / L trace element solution (54.4 g / L ferric ammonium citrate, 9.8 g / L MnCl₂×4H₂O, 1.6 g / L CoCl₂×6H₂O, 1 g / L CuCl₂×2H₂O, 1.9 g / L H₃BO₃, 9 g / L ZnSO₄×7H₂O, 1.1 g / L Na₂MoO₄ 2H₂O, 1.5 g / L Na₂SeO₃, 1.5 g / L...). NiSO4×6H2O was used, containing 2% (v / v) glycerol, 2% (m / v) sterile filtered sucrose, or 2% sucrose hydrolysate as carbon sources. Additionally, 15 mM lactose was added to generate an acceptor substrate for the production of 2'-fucosylated lactose. Cultures were inoculated to an OD of 0.1 and culture was stopped after 26 hours. To quantify 2'-FL in the culture medium, HPLC analysis was performed using a refractive index detector (RID-10A) connected to an HPLC system (Shimadzu, Germany) and a Waters XBridge Amide 3.5 μm (250 x 4.6 mm) column (Eschborn, Germany). Isocratic elution was performed at 35°C using 30% A: 50% (v / v) ACN dissolved in ddH₂O, 0.1% (v / v) NH₄OH, and 70% B: 80% (v / v) ACN dissolved in ddH₂O, 0.1% (v / v) NH₄OH as the eluent, at a flow rate of 1.4 ml / min. -1The culture supernatant was sterilely filtered (0.22 μm pore size) and removed by solid-phase extraction on an ion-exchange matrix (Strata ABW, Phenomenex). 10 μl of sample was loaded onto the column, and the 2'-fucosylated lactose concentration was calculated based on the standard curve. The productivity of the engineered strain during growth on glycerol was set to 100%. As shown in Table 3, the highest 2'-FL yield was achieved when sucrose hydrolysis products were provided as both a carbon and energy source.

[0150] glycerin sucrose Sucrose hydrolysis products Relative 2'-FL production 100％ 199％ 482％

[0151] Table 3: Relative 2'-FL yield of the *E. coli* strain described in Example 3 during cultivation using glycerol, sucrose, or sucrose hydrolysate as carbon and energy sources. Productivity on glycerol was set to 100%.

[0152] Example 4 - Total fermentation of 2'-fucosylated lactose by engineered Escherichia coli strains on a mixed monosaccharide feedstock

[0153] By overexpressing enzymes used for the de novo synthesis of GDP-fucose (ManB, ManC, Gmd, WcaG), the following genes were used: wbgL, a 2'-fucosyltransferase gene from *E. coli* O126; yberc0001_9420, a sugar efflux transporter gene from *Yersinia poconiosis* ATCC 43970; glf, a glucose-promoting factor gene from *Fermentomonas motilityis*; galTpm1141, a β-galactosyltransferase gene from *Pasteurella multocida* (GenBank: AEC04686); and the *E. coli* genes galE and pgm, which encode UDP-glucose 4-epimerase and phosphoglucose mutase, respectively. Further genetic engineering revealed the genotype pfkA. - ,lacZ,fuclK - wcaJ - , glk - gcd - pstG -*Escherichia coli* strain BL21(DE3) was used. Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000)). Heterologous genes were integrated into the genome via transposition. Linear DNA fragments were integrated using the EZ-Tn5™ transposase (Epicentre, USA) or a highly active C9- mutant of the sailor transposase Himar1 (Proc. Natl. Acad. Sci. 1999, USA 96: 11428-11433) was used for transposition. These genes were codon-optimized for expression in *E. coli* and were synthesized by GenScript.

[0154] The *E. coli* strain cultured at 30°C in a 3L fermenter (New Brunswick, Edison, USA) was started with 1000 mL of mineral salt medium containing 7 g / L of water. -1 NH4H2PO4, 7 g·L -1 K2HPO4, 2g·L - 1 KOH, 0.3 g·L -1 Citric acid, 2 g·L -1 MgSO4×7·H2O and 0.015 g·L -1 Add CaCl2×6·H2O, and replenish 1 mL / L. -1 Trace element solution (54.4 g·L) -1 Ferric ammonium citrate, 9.8 g·L -1 MnCl2×4·H2O, 1.6 g·L -1 CoCl2×6·H2O, 1g·L -1 CuCl2×2·H2O, 1.9 g·L -1 H3BO3, 9g·L -1 ZnSO4×7·H2O, 1.1 g·L -1 Na₂MoO₄×2·H₂O, 1.5 g·L -1 Na2SeO3, 1.5 g·L -1 NiSO4×6·H2O was prepared, containing 2% (m / v) hydrolyzed sucrose as a carbon source. Culture was initiated by adding 2.5% (v / v) inoculum from a preculture grown in the same medium. The end of the batch phase was characterized by a rise in dissolved oxygen levels. Immediately after the end of the batch phase, a carbon feed consisting of fully hydrolyzed sucrose was applied, supplemented with 2 g·L⁻¹. -1 MgSO4×7·H2O, 0.015 g·L -1 CaCl2×6·H2O and 1mL·L-1 Trace element solution. Feed rate: 12.0-15.0 mL / L -1 ·h -1 Refer to the initial volume. Maintain inflation at 3 L / min. -1 Dissolved oxygen was maintained at 20-30% saturation by controlling the stirring rate. The pH was maintained at 6.7 by adding 25% ammonia solution. The culture lasted for 86 hours, and a large amount of 2'-FL was generated in the culture supernatant. sequence list <110> Janiwin Biotechnology LLC <120> Carbohydrate production through fermentation using microbial cells from a mixture of raw materials. <130> CP1210732P <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 1395 <212> DNA <213> Escherichia coli <400> 1 atgcctgacg ctaaaaaaca ggggcggtca aacaaggcaa tgacgttttt cgtctgcttc 60 cttgccgctc tggcgggatt actctttggc ctggatatcg gtgtaattgc tggcgcactg 120 ccgtttattg cagatgaatt ccagattact tcgcacacgc aagaatgggt cgtaagctcc 180 atgatgttcg gtgcggcagt cggtgcggtg ggcagcggct ggctctcctt taaactcggg 240 cgcaaaaaga gcctgatgat cggcgcaatt ttgtttgttg ccggttcgct gttctctgcg 300 gctgcgccaa acgttgaagt actgattctt tcccgcgttc tactggggct ggcggtgggt 360 gtggcctctt ataccgcacc gctgtacctc tctgaaattg cgccggaaaa aattcgtggc 420 agtatgatct cgatgtatca gttgatgatc actatcggga tcctcggtgc ttatctttct 480 gataccgcct tcagctacac cggtgcatgg cgctggatgc tgggtgtgat tatcatcccg 540 gcaattttgc tgctgattgg tgtcttcttc ctgccagaca gcccacgttg gtttgccgcc 600 aaacgccgtt ttgttgatgc cgaacgcgtg ctgctacgcc tgcgtgacac cagcgcggaa 660 gcgaaacgcg aactggatga aatccgtgaa agtttgcagg ttaaacagag tggctgggcg 720 ctgtttaaag agaacagcaa cttccgccgc gcggtgttcc ttggcgtact gttgcaggta 780 atgcagcaat tcaccgggat gaacgtcatc atgtattacg cgccgaaaat cttcgaactg 840 gcgggttata ccaacactac cgagcaaatg tgggggaccg tgattgtcgg cctgaccaac 900 gtacttgcca cctttatcgc aatcggcctt gttgaccgct ggggacgtaa accaacgcta 960 acgctgggct tcctggtgat ggctgctggc atgggcgtac tcggtacaat gatgcatatc 1020 ggtattcact ctccgtcggc gcagtatttc gccatcgcca tgctgctgat gtttattgtc 1080 ggttttgcca tgagtgccgg tccgctgatt tgggtactgt gctccgaaat tcagccgctg 1140 aaaggccgcg attttggcat cacctgctcc actgccacca actggattgc caacatgatc 1200 gttggcgcaa cgttcctgac catgctcaac acgctgggta acgccaacac cttctgggtg 1260 tatgcggctc tgaacgtact gtttatcctg ctgacattgt ggctggtacc ggaaaccaaa 1320 cacgtttcgc tggaacatat tgaacgtaat ctgatgaaag gtcgtaaact gcgcgaaata 1380 ggcgctcacg attaa 1395 <210> 2 <211> 1422 <212> DNA <213> Zymomonas mobilis <400> 2 atgagttctg aaagtagtca gggtctagtc acgcgactag ccctaatcgc tgctataggc 60 ggcttgcttt tcggttacga ttcagcggtt atcgctgcaa tcggtacacc ggttgatatc 120 cattttattg cccctcgtca cctgtctgct acggctgcgg cttccctttc tgggatggtc 180 gttgttgctg ttttggtcgg ttgtgttacc ggttctttgc tgtctggctg gattggtatt 240<00​​ggtgctgcgt taaccgaaaa attatttgga accggtggtt cggctttaca aattttttgc 360 tttttccggt ttcttgccgg tttaggtatc ggtgtcgttt caaccttgac cccaacctat 420 attgctgaaa ttcgtccgcc agacaaacgt ggtcagatgg tttctggtca gcagatggcc 480 attgtgacgg gtgctttaac cggttatatc tttacctggt tactggctca tttcggttct 540 atcgattggg ttaatgccag tggttggtgc tggtctccgg cttcagaagg cctgatcggt 600 attgccttct tattgctgct gttaaccgca ccggatacgc cgcattggtt ggtgatgaag 660 ggacgtcatt ccgaggctag caaaatcctt gctcgtctgg aaccgcaagc cgatcctaat 720 ctgacgattc aaaagattaa agctggcttt gataaagcca tggacaaaag cagcgcaggt 780 ttgtttgctt ttggtatcac cgttgttttt gccggtgtat ccgttgctgc cttccagcag 840 ttagtcggta ttaacgccgt gctgtattat gcaccgcaga tgttccagaa tttaggtttt 900 ggagctgata cggcattatt gcagaccatc tctatcggtg ttgtgaactt catcttcacc 960 atgattgctt cccgtgttgt tgaccgcttc ggccgtaaac ctctgcttat ttggggtgct 1020 ctcggtatgg ctgcaatgat ggctgtttta ggctgctgtt tctggttcaa agtcggtggt 1080 gttttgcctt tggcttctgt gcttctttat attgcagtct ttggtatgtc atggggccct 1140 gtctgctggg ttgttctgtc agaaatgttc ccgagttcca tcaagggcgc agctatgcct 1200 atcgctgtta ccggacaatg gttagctaat atcttggtta acttcctgtt taaggttgcc 1260 gatggttctc cagcattgaa tcagactttc aaccacggtt tctcctatct cgttttcgca 1320 gcattaagta tcttaggtgg cttgattgtt gctcgcttcg tgccggaaac caaaggtcgg 1380 agcctggatg aaatcgagga gatgtggcgc tcccagaagt ag 1422 <210> 3 <211> 1248 <212> DNA <213> Escherichia coli <400> 3 atggcactga atattccatt cagaaatgcg tactatcgtt ttgcatccag ttactcattt 60 ctcttttta tttcctggtc gctgtggtgg tcgttatacg ctatttggct gaaaggacat 120 ctagggttga cagggacgga attaggtaca cttattcgg tcaaccagtt taccagcatt 180 ctatttatga tgttctacgg catcgttcag gataaactcg gtctgaagaa accgctcatc 240 tggtgtatga gtttcatcct ggtcttgacc ggaccgttta tgatttacgt ttatgaaccg 300 ttactgcaaa gcaatttttc tgtaggtcta attctggggg cgctattttt tggcttgggg 360 tatctggcgg gatgcggttt gcttgatagc ttcaccgaaa aaatggcgcg aaattttcat 420 ttcgaatatg gaacagcgcg cgcctgggga tcttttggct atgctattgg cgcgttcttt 480 gccggcatat tttttagtat cagtccccat atcaacttct ggttggtctc gctatttggc 540 gctgtattta tgatgatcaa catgcgtttt aaagataagg atcaccagtg cgtagcggca 600 gatgcgggag gggtaaaaaa agaggatttt atcgcagttt tcaaggatcg aaacttctgg 660 gttttcgtca tatttattgt ggggacgtgg tctttctata acatttttga tcaacaactt 720 tttcctgtct tttattcagg tttattcgaa tcacacgatg taggaacgcg cctgtatggt 780 tatctcaact cattccaggt ggtactcgaa gcgctgtgca tggcgattat tcctttcttt 840 gtgaatcggg tagggccaaa aaatgcatta cttatcggag ttgtgattat ggcgttgcgt 900 atcctttcct gcgcgctgtt cgttaacccc tggattatttt cattagtgaa gttgttacat 960 gccattgagg ttccactttg tgtcatatcc gtcttcaaat acagcgtggc aaactttgat 1020 aagcgcctgt cgtcgacgat ctttctgatt ggttttcaaa ttgccagttc gcttgggatt 1080 gtgctgcttt caacgccgac tgggatactc tttgaccacg caggctacca gacagttttc 1140 ttcgcaattt cgggtattgt ctgcctgatg ttgctatttg gcattttctt cttgagtaaaa 1200 aaacgcgagc aatagttat ggaaacgcct gtaccttcag caatatag 1248 <210> 4 <211> 735 <212> DNA <213> Leuconostoc pseudomesenteroides <400> 4 atggcacaaa acgcgcaaca tcataatccg cgaagcattt caatgagcaa atcacttatg 60 ttttttgcca tctcattgat tttaaatgcg atgggaaatg ttttgacgct cgtcacagct 120 tcacatataa aacccgcttt tttgggatca gcttattgga ctgctgcaga ggctaatcta 180 ggtcaagctt tattaggaaa taattcactt gtcttgtttt gggcattttt agttcttggc 240 atgcttattt cattcctgaa tgcgttatta atgaaaaagt tagattggca tcgtatcatt 300 ggtaatttct tgtttatgtt accattttca atttttattc aatggttttc aaatattttc 360 aatcaaatta tgccaaatgc taattcagtg gcagcaactg tgttatatac agtaattaat 420 tttattggtg ttggcttgat cgccgttgct atttcgattt atcaacgtgt gaatttagtg 480 ttacaccctg ccgatgattt gatgcaaatt ttacgtttca aatattttca cgggtcggct 540 ttcaaggcta tgtgggcgtc ctatattccg ccaacgattt ttgcaattat tgcctttgtg 600 atcactttcc ctaatttgta taatttcggg ttaggaatta tttttgcatt cttattccag 660 ggtggcatca caggaatcgc ggacaagtac gtctttaaga atttaaagca tcaggcaata 720 gatgttggta attaa 735 <210> 5 <211> 1254 <212> DNA <213> Escherichia coli <400> 5 atgtactatt taaaaaac aaacttttgg atgttcggtt tattctttt cttttacttt 60 tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120 agcaaaagtg attack tatttttgcc gctatttctc tgttctcgct attack ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240 accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa tacaacattt tagtagtc gattgttggt ggtatttatc tagcttttg ttttaacgcc ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480 atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540 gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600 gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720 caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta tttggctacg they gggcgat cttaacgcct cgattatgtt ctttgcgcca ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900 gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960 ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020 tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttct taagcaactg 1080 gcgatgattt ttgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140 gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200 agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc ttaa 1254 <210> 6 <211> 1317 <212> DNA <213> Escherichia coli <400> 6 atgggaaaca catcaataca aacgcagagt taccgtgcgg tagataaaga tgcagggcaa 60 agcagaagtt acattattcc attcgcgctg ctgtgctcac tgttttttct ttgggcggta 120 gccaataacc ttaacgacat tttattacct caattccagc aggcttttac gctgacaaat 180 ttccaggctg gcctgatcca atcggccttt tactttggtt attcattat cccaatccct 240 gctgggatat tgatgaaaaa actcagttat aaagcaggga ttattaccgg gttattttta 300 tatgccttgg gtgctgcatt attctggccc gccgcagaaa taatgaacta caccttgttt 360 ttagttggcc tattattat tgcagccgga ttaggttgtc tggaaactgc cgcaaaccct 420 tttgttacgg tattagggcc ggaaagtagt ggtcacttcc gcttaaatct tgcgcaaaca 480 tttaactcgt ttggcgcaat tatcgcggtt gtctttgggc aaagctctat tttgtctaac 540 gtgccacatc aatcgcaaga cgttctcgat aaaatgtctc cagagcaatt gagtgcgtat 600 aaacacagcc tggtattatc ggtacagaca ccttatatga tcatcgtggc tatcgtgtta 660 ctggtcgccc tgctgatcat gctgacgaaa ttcccggcat tgcagagtga taatcacagt 720 gacgccaaac aggatcgtt ctccgcatcg ctttctcgcc tggcgcgtat tcgccactgg 780 cgctgggcgg tattagcgca attctgctat gtcggcgcac aaacggcctg ctggagctat 840 ttgattcgct acgctgtaga agaaattcca ggtatgactg caggctttgc cgctaactat 900 ttaaccggaa ccatggtgtg cttctttatt ggtcgtttca ccggtacctg gctcatcagt 960 cgcttcgcac cacacaaagt cctggccgcc tacgcattaa tcgctatggc actgtgcctg 1020 atctcagcct tcgctggcgg tcatgtgggc ttaatagccc tgactttatg cagcgccttt 1080 atgcgattc agtacccaac aatcttctcg ctgggcatta agaatctcgg ccaggacacc 1140 aaattggtt cgtccttcat cgttatgacc attattggcg gcggtattgt cactccggtc 1200 atgggtttg tcagtgacgc ggcgggcaac atccccactg ctgaactgat ccccgcactc 1260 tgcttcgcgg tcatctttat ctttgcccgt ttccgttctc aaacggcaac taactga 1317 <210> 7 <211> 1491 <212> DNA <213> Escherichia coli <400> 7 atgagtacta caacccagaa tatcccgtgg tatcgccatc tcaaccgtgc acaatggcgc 60 gcatttccg ctgcctggtt gggatatctg cttgacggtt ttgatttcgt tttaatcgcc 120 ctggtactca ccgaagtaca aggtgaattc gggctgacga cggtgcaggc ggcaagtctg 180 atctctgcag cctttatctc tcgctggttc ggcggcctga tgctcggcgc tatgggtgac 240 cgctacgggc gtcgtctggc aatggtcacc agcatcgttc tcttctcggc cgggacgctg 300 gcctgcggct ttgcgccagg ctacatcacc atgtttatcg ctcgtctggt catcggcatg 360 gggatggcgg gtgaatacgg ttccagcgcc acctatgtca ttgaaagctg gccaaaacat 420 ctgcgtaaca aagccagtgg ttttttgatt tcaggcttct ctgtgggggc cgtcgttgcc 480 gctcaggtct atagcctggt ggttccggtc tggggctggc gtgcgctgtt ctttatcggc 540 attttgccaa tcatctttgc tctctggctg cgtaaaaaca tcccggaagc ggaagactgg 600 aaagagaaac acgcaggtaa agcaccagta cgcacaatgg tggatattct ctaccgtggt 660 gaacatcgca ttgccaatat cgtaatgaca ctggcggcgg ctactgcgct gtggttctgc 720 ttcgccggta acctgcaaaa tgccgcgatc gtcgctgttc ttgggctgtt atgcgccgca 780 atctttatca gctttatggt gcagagtgca ggcaaacgct ggccaacggg cgtaatgctg 840 atggtggtcg tgttgtttgc tttcctctac tcatggccga ttcaggcgct gctgccaacg 900 tatctgaaaa ccgatctggc ttataacccg catactgtag ccaatgtgct gttctttagt 960 ggctttggcg cggcggtggg atgctgcgta ggtggcttcc tcggtgactg gctgggaacc 1020 cgcaaagcgt acgtttgtag cctgctggcc tcgcagctgc tgattattcc ggtatttgcg 1080 attggcggcg caaacgtctg ggtgctcggt ctgttactgt tcttccagca aatgcttgga 1140<000一切, caagggatcg ccgggatctt accaaaactg attggcggtt atttcgatac cgaccagcgt 1200 gcagcgggcc tgggctttac ctacaacgtt ggcgcattgg gcggtgcact ggccccaatc 1260 atcggcgcgt tgatcgctca acgtctggat ctgggtactg cgctggcatc gctctcgttc 1320 agtctgacgt tcgtggtgat cctgctgatt gggctggata tgccttctcg cgttcagcgt 1380 tggttgcgcc cggaagcgtt gcgtactcat gacgctatcg acggtaaacc attcagcggt 1440 gccgtgccgt ttggcagcgc caaaaacgat ttagtcaaaa ccaaaagtta a 1491 <210> 8 <211> 1278 <212> DNA <213> Xanthomonas campestris <400> 8 atgactaccg ccaggcccgc aaatcccgtt gtctcgatcg ccatcgtcgg cgtgctgttt 60 ttcatcatcg gctttttcac ctggatcaac gggccgctga tcaccttcgt gcggctggcg 120 ttcgacctca acgaggtcaa tgcgttcctg gtgctgatgg tgttctacct gtcgtacttc 180 ttcctggcgc tgccctcgtc atggatcctc aagcgcaccg gcatgaaaaa aggcctggcg 240 ctgagtctgg tggtgatggc gctgggcgcc gcagcattcg ggcaattcgc tacgcaacgc 300 tggtatccgg gcgcactggc cggcatgttc gtgatcggta gcggcctggc gttgttgcag 360 accgcgatca acccatacat cagtattctc gggccaatcg aaagtgcggc gcggcgcatc 420 gcgctgatgg gcatctgtaa caagattgcc ggcatcctgg cgccgatcct gatcggctcg 480 ctggtgctgc atggcatcgg cgatctctcc acgcaggtgg ccgcggccga tgcggcgacc 540 aagcagcaac tgctcaacgc gtttgccgcc aagatccatg caccatatct ggtgatgtcc 600 ggcgtgttgc tggtgctggc ggtgggcgtg ttgttctcgc cgctgcccga actcaaggcc 660 tccgaagcca atgccacgcc gggcagcggt ggcgcggcgc agaagtccag catcttccag 720 ttcccgcatc tgtggctggg cgtgttgtgc ctgttcgtgt atgtcggtgt ggaagtgatg 780 gccggcgatg ccatcggcac ttacggacac ggcttcaatt tgccgctgga cagcaccaag 840 ctgttcacct cctacacgct gggcgcaatg ttgctgggct atatcgccgg cctggtgctg 900 attccgcggg tgatttcgca ggcgcgctac ctgagcgtgt ctgcgctgct gggcgtgctg 960 ttctcgctgg gggcgctgtt cacccacggc tatgtgtcgg tggggttcgt ggccgcgctc 1020 ggttttgcca acgccatgat gtggccggcg atctttccgc tggccatccg cggcctgggc 1080<000​​​​​​​​​​​​​​​​​​​​agcgcaaccg gtgcggcggc caccgcaatc gaaaccatcg aaaccggcgt ggccggagtg 120 gcgggcgcgg ccacaaacgc ggcagccaac gcaatcgagg acctcgaagc cgccgaatcg 180 cacggcttct ccacgcgctt cccgctcaac agcgcattca tcttcacctt cggcgcgctc 240 ggcggcatgc tgttcggttt cgacaccggc atcatctccg gcgcctcccc gcttatcgaa 300 tcggacttcg gcctgagcgt ctctcagacc ggcttcatca cctcctcggt gctgatcggc 360 tcgtgcgcag gcgcgctgtc gatcggcgca ctgtctgacc ggttcggccg caagaagctg 420 ctcatcgtct ccgcgctgct gttcctgctc ggctcaggcc tgtgcgcctc ctccaccgga 480 ttcgcgatga tggtgtgcgc ccgcatcatc ctgggtctcg ccgtcggcgc ggcctccgcc 540 ctgaccccgg cgtacttggc cgaactggcg ccgaaggagc gtcgcggctc actgtccacg 600 ctgttccagc tcatggtcac cttcggcatt ctgctggcct acgcctccaa cctcggattc 660 ctgaaccaca acctcttcgg catccgcgac tggcgctgga tgctcggttc ggcgctggtg 720 ccggccgcct tgttgctgct cggcggcctg ttgctgcccg aatccccgcg ttatctggtg 780 aacaagggcg acacccgcaa cgccttcaaa gtgcttacgc tgattcgcaa ggacgtggat 840 cagacccagg tgcagattga gctggacgaa atcaaggccg tggccgcaca ggacaccaag 900 ggtggtgtgc gcgaactgtt ccgtatcgct cgtccggcgc tggtggccgc catcggtatc 960 atgctgttcc agcagctcgt gggcatcaac tcggtgatct acttcctgcc gcaggtgttc 1020 atcaagggct tcggcttccc tgaaggcgac gcgatctggg tgtcggtggg catcggcgtg 1080 gtgaacttcg tgagcaccat cgtggccacg cttatcatgg atcgtttccc gcgcaagggc 1140 atgctgatct tcggttccat cgtgatgacc gtttcgctcg cggtgctcgc cgtgatgaac 1200 ttcgtgggcg acgtggccgt gctggcagtg ccgacgatga ttctcatcgc tttctatatc 1260 ctcggctttg cggtctcgtg gggcccgatc gcctgggtgc ttatcggcga gatcttcccg 1320 ctgagcgtgc gcggcatcgg ctcatccttc ggctcggcgg cgaactggct gggcaacttc 1380 atcgtctccc agttcttcct cgtgctgctc gatgcgttcg gcaacaatgt gggcggcccg 1440 ttcgcgattt tcggcgtgtt ctcggccctg tccatcccgt tcgtgctgcg cttggtgccc 1500 gagaccaagg gcaagtcgct ggaggaaatc gagaaggaaa tgaccaagcg ctag 1554 <210> 10 <211> 1206 <212> DNA <213> Bacillus subtilis <400> 10 atgttaagag ggacatattt atttggatat gctttcttt ttacagtagg tattatccat 60 atatcaacag ggagtttgac accattttta ttagaggctt ttaacaagac aacagatgat 120 atttcggtca taatcttctt ccagtttacc ggatttctaa gcggagtatt aatcgcacct 180 ttaatgatta agaaatacag tcattttagg acacttactt tagctttgac aataatgctt 240 gtagcgttaa gtatcttttt tctaaccaag gattggtatt atattattgt aatggctttt 300 ctcttaggat atggagcagg cacattagaa acgacagttg gttcatttgt tattgctaat 360 ttcgaaagta atgcagaaaa aatgagtaag ctggaagttc tctttggatt aggcgcttta 420 tctttcccat tattaattaa ttccttcata gatatcaata actggttttt accatattac 480 tgtatattca cctttttatt cgtcctattc gtagggtggt taattttctt gtctaagaac 540 cgagagtacg ctaagaatgc taaccaaca gtgaccttttc cagatggagg agcattcaa 600 tactttatag gagatagaaa aaatcaaag cattaggct ttttgtatt ttcgctttc 660 ctatatgctg gattgaaac aaattttgcc aactttttac cttcaatcat gataaaccaa 720 bōkatgaac aaattagtct tataagtgtc tccttttttct gggtaggt catcatagga 780 agaatattga ttggttcgt aagtagaagg cttgatttt ccaatacct tctttttagc 840 tgtagttgtt taattgtttt gttgattgcc ttctcttata taagtaaccc atacttcaa 900 ttgagtgta cattttgat tggcctaagt atagcgggga tatttcccat tgctttaaca 960 ctagcatcaa tcattattca gaagtacgtt gacgaagtta caagtttatt tattgccctcg 1020 gcaagtttcg gaggagcgat catctctttc ttaattggat ggagtttaaa ccaggatacg 1080 atcttattaa ccatgggaat atttacaact atggcggtca ttcttagg tattctctgta 1140 agattagga gaacataac agagaccct atttcacttg aaaaaaagc atcaaaaca 1200 Chapter 1206 <210> 11 <211> 1593 <212> DNA <213> Vibrio parahaemolyticus <400> 11 atggtcttcg ccatttatgt cgcaattatc attggggtcg gactttgggt atctcgtgat 60 aaaaaaggca ctcagaaaag tacggaagat tatttcttgg cgggaaaatc tttgccttgg 120 tgggctgtcg gtgcttcgct aattgctgca aatatttctg cggaacaatt tataggaatg 180 tctggttcag gctattcaat tggcttggct atcgcatctt atgaatggat gtcggcaata 240 acattgatta ttgttggtaa gtactttcta cctattttca ttgaaaaagg aatctatacc 300 attcctgaat ttgttgaaaa acgcttcaat aaaaaactaa aaacaatttt ggccgttttt 360 tggatttcct tgtacatttt tgtaaaccta acttcagtac tgtatttagg tggtttggct 420 ctcgaaacca ttttgggtat tccgttgatg tactcaattc taggtcttgc gctgtttgcg 480 ttggtgtact caatttatgg tggtttatcg gcagtagtat ggaccgatgt catccaagtg 540 ttcttcttag ttttgggtgg ttttatgact acctacatgg cagtgagctt tattggtggt 600 acggacggtt ggttcgctgg ggtgtctaaa atggtcgatg cagctcctgg ccactttgag 660 atgatcttgg atcaaagtaa tccacaatac atgaaccttc ctggtattgc cgtattaatt 720 ggtggtcttt gggtagcaaa cttatattac tggggcttta accagtacat tattcaaaga 780 acgcttgctg caaaatcagt atcggaagct cagaaaggta ttgtttttgc agcgtttttg 840 aaacttatcg ttccgtttct cgtggtattg ccaggtattg ccgcttacgt tattacttcg 900 gacccacaac taatggcaag ccttggtgat attgcagcaa caaaccttcc aagtgctgct 960 aatgcggata aagcataccc ttggctaact cagttcttgc ctgttggtgt taaaggtgtt 1020 gtttttgcgg ctcttgctgc tgcaattgtt tcttcactag catcaatgct taactcaaca 1080 gccactatct tcactatgga tatttacaaa gagtatatct ctcctgactc aggtgaccac 1140 aagttggtga atgttgggcg tactgcagct gtggtggcac taattattgc ttgcctaatt 1200 gccccaatgt taggtggtat tggccaagca ttccaataca tccaagaata tacaggttta 1260 gttagccctg gtattttggc tgtattctta cttggcttat tctggaagaa aacaaccagt 1320 aaaggggcta ttatggtgt agtagcatca ataccatttg ccttgttctt gaaatttatg 1380 ccactttcca tgccatttat ggatcaaatg ctatacacat tattgtttac aatggttgtt atcgcattta caagtttgag cacatcaatt aatgatgatg atcctaaagg tattagtgtt acatcatcga tgtttgtaac agatcgaagc tttaatatcg ctgcttacgg cataatgatt gttttggcag tgttatatac attgttctgg taa <210> 12 <211> 1476 <212> DNA <213> Liquid(Escherichia coli) <400> 12 atgaataccc agtataattc cagttatata ttttcgatta ccttagtcgc tacattaggt ggtttattat ttggctacga caccgccgtt atttccggta ctgttgagtc actcaatacc gtctttgttg ctccacaaaa cttaagtgaa tccgctgcca actccctgtt agggttttgc gtggccagcg ctctgattgg ttgcatcatc ggcggtgccc tcggtggtta ttgcagtaac 240 cgcttcggtc gtcgtgattc acttaagatt gctgctgtcc tgttttttat ttctggtgta 300 360. ggttctgcct ggccagaact tggttttacc tctataaacc cggacaacc tgtgcctgtt tatctggcag gttatgtccc ggaatttgtt atttatcgca ttattggcgg tattggcgtt 420 ggtttagcct caatgctctc gccaatgtat attgcggaac tggctccagc tcatattcgc 480 gggaaactgg tctcttttaa ccagtttgcg attattttcg ggcaactttt agtttactgc 540 gtaaactatt ttattgcccg ttccggtgat gccagctggc tgaatactga cggctggcgt 600 tatatgtttg cctcggaatg tatccctgca ctgctgttct taatgctgct gtataccgtg 660 ccagaaagtc ctcgctggct gatgtcgcgc ggcaagcaag aacaggcgga aggtatcctg 720 cgcaaaatta tgggcaacac gcttgcaact caggcagtac aggaaattaa acactccctg 780 gatcatggcc gcaaaaccgg tggtcgtctg ctgatgtttg gcgtgggcgt gattgtaatc 840 ggcgtaatgc tctccatctt ccagcaattt gtcggcatca atgtggtgct gtactacgcg 900 ccggaagtgt tcaaaacgct gggggccagc acggatatcg cgctgttgca gaccattatt 960 gtcggagtta tcaacctcac cttcaccgtt ctggcaatta tgacggtgga taaatttggt 1020 cgtaagccac tgcaaattat cggcgcactc ggaatggcaa tcggtatgtt tagcctcggt 1080 accgcgtttt acactcaggc accgggtatt gtggcgctac tgtcgatgct gttctatgtt 1140 gccgcctttg ccatgtcctg gggtccggta tgctgggtac tgctgtcgga aatcttcccg 1200. aatgctattc gtggtaagc gctggcaatc gcggtggcgg cccagtggct ggcgaactac ttcgtctcct ggaccttccc gatgatggac aaaaactcct ggctggtggc ccatttccac 1320 aacggtttct cctactggat ttacggttgt atgggcgttc tggcagcact gtttatgtgg 1380. aaatttgtcc cggaaccaa aggtaaaacc cttgaggagc tggaagcgct ctgggaaccg 1476. aactgctacg ctgtaa <210> 13 <211> 1395 <212> DNA <213> Bacillus subtilis (Bacillus subtilis) <400> 13 attack ctccaactca attack aatgttcctg ccattcaatg ggatttgtca ttttgatctc atgtgcggcg gggcttggcg gcttattgta tggctatgac 120 acggcagtga tttctggcgc catcggtttt ctgaaagatt tatacagcct gagtccgttt atggagggac ttgtcatttc aagcattatg attggaggag ttgtgggcgt cgggatatcc 240 ggatttttaa gtgacagatt cggccggaga aaaattttaa tgacagccgc tttgttattt gcgatatcag caatcgtttc agcgctttct caacgtgt ccaccttaat cattgcaagg 360 attatcgggg ggctgggaat cgggatggggc tcatcgctct ctgttacgta tattacagaa 420 gcggcaccgc ccgctatacg cggaagttta tcttcgttat atcagctctt tacgatactg 480 ggtatttccg cacatactt tattaatcta gctgtgcagc ggtccggaac atacgaatgg 540 ggcgtgcaca ccggctggag atggatgctt gcttatggaa tggtgccatc cgtcatttt 600 ttccttgtcc tgctcgtcgt cccggaagt ccgagatggc tggcgaagc gggcaaaca 660 aatgaagctt windowacct gatacgtattt aatgagaaa ctgttgcaaa agagaatta 720 aagaacattg agaactcttt aaaaatagaa caaatggggt cgctctccca gctgtttaag 780 ccgggtca gaaaggcgct tgtcattgga atcctgctgg cgctgtta ccaagtcatc 840 ggcatgaacg cgattactta ctacgggccg gaatcttta aaatgatggg attcgggcaa 900 aacgccggat ttgtgacgac ttgtatcgtc ggggttgtag aagttattt taccgttatt 960 gcggtgttgc tgattgataa agtcggacga aaaaaactga tgtccatcgg ttctgctttt 1020 atggctattt ttatgattt aatcggggacg tcgttttatt ttgagttaac aagcgggatc 1080 atgatgatcg tccttatatt aggttttgtc gctgctttct gtgtctcggt cggaccgatc 1140 acatggatta tgattctga aatcttcccg aaccatctgc gtgcgcgggc cgcgggcatt 1200 gcgaccatct tttatgggg agcaaactgg gcgatcggac agtttgtgcc aatgatgatc 1260 gattctttcg ggctcgccta tacattttgg atctttgcgg tgattaacat cctttgtttc 1320 ctgtttgtcg ttacgatctg tccagaaacg aagaacaaat cgctcgagga aattgaaaag 1380 ctttggataa aatga 1395 <210> 14 <211> 4393 <212> DNA <213> Klebsiella pneumoniae (Klebsiella pneumoniae) <400> 14 atgtataaaa aacggaagtt agccattctt attgctttgc taaccggcac cgccgccgcc 60 catgggcaga cagacctgaa cagcattgaa gcgcgtctcg ccgccctgga aaaacgcctg 120 caggacgccg agacccgc cagcactgcc gaaagccgcg ccgcctcagc ggagcagaaa 180 gttcagcagt taacccagca gcagcagcaa acccaggcca ccacccagca ggtggccagg 240 cgcaccactc aactggaaga aaaagccgaa cggcccggcg gctttgagtt ccatggctat 300 gcgcgttccg gggtgatcat gaacgactcg gccgccagta ccaaatccgg cgcttatatg 360 acccccgccg gggagaccgg cggcgccatt ggtcgcctgg gcaaccaggc cgacacctat 420 gtggaaatga acctcgaaca taaacagacc ctggacaacg gggcgaccac ccgtttcaaa 480 gtgatggtgg ccgacggaca gaccacctat aacgactgga cggcaagcag cagcgacctg 540 aacgtacgcc aggcgttcgt cgagctgggc aatctgccga ccttcgaagg cccgttcaaa 600 ggctcgaccc tgtgggccgg gaaacgcttt gaccgcgaca acttcgacat ccactggatt 660 gactcggatg tggtgttcct cgccgggacc ggcggcggga tctacgacgt gaaatggaac 720 gacagcctgc gcagcaactt ctcgttatac ggccgcaact ttggcgatat cgccgacagc 780 agcaacagcg tgcagaacta tatcgtcagc atgaataact ttgccggccc ggtgcagatg 840 atggtcagcg ggatgcgggc gaaagataat gacgaccgcc aggacgcgaa cggcaatctg 900 gtgaaaggcg atgccgctaa caccggggtt catgccctgc tgggcctgca caatgagagc 960 ttctatggcc tgcgcgacgg gaccagcaaa acggccctgc tgtacggcca cgggctgggc 1020 gccgaggtta aaggcatcgg ctccgacggc gcgctgcgcc cgggggccaa tacctggcgc 1080 ttcgccagct atggcactac gccgctgagc gatcgctggt ttattgcccc ggccgtgctg 1140 gcgcagagca gtaaagatcg tttgcgat ggcgacagct atcagtgggc caccctcaac 1200 ctgcgtctga ttcaggaagt gacgcagaac ttcgccctcg cctgggaggg cagctatcag 1260 tacatggatc tgcagcctga aggctacaac gatcgccatg cggtcaatgg cagcttctac 1320 aagctgacct tcgccccgac cttcaaggtg ggcagcatcg gcgacttctt ctcgcggccg 1380 gagatccgct tctatacatc gtggatggac tggagcaaaa aactggacaa ctacgccaac 1440 gatgacgcgt taggcagcaa cggattcaaa tcgggcggcg aatggtcgtt cggtatgcaa 1500 atggagacct ggttctgacg gccaccgggg cgacagggta aataacacat aatataagg 1560 ttcgcggcgc ctgccacggc tggcgccgcc cacgccatat catcatgcat ttagaggggta 1620 ctatggattt tgaacagatt tcccgctcac tgcttcccct gctgggcggc aaggaaaata 1680 tcgccagcgc cgcgcactgc gccacccgcc tgcggctggt gctggtcgac gacgcgctcg 1740 ccgatcagca ggcgattggc aaaatcgacg gggtgaaagg ctgctttcgc aatgccggac 1800 agatgcagat catcttcggc accggggtgg tcaataaagt ctatgccgcc tttatccagg 1860 ccgcaggcat cagcgaatcg agcaaatccg aagccgccga cctggcggcg aaaaagctga 1920 acccgttcca gcgcatcgcc cgcctgctgt ccaacatctt cgtgccgatt attccggcca 1980 tcgtcgcctc cggcctgctg atgggcctgc tggggatggt gaaaacctac ggttgggtcg 2040 acccgagcaa cgctctctat atcatgctgg atatgtgcag ttcggcggcg tttatcattc 2100 tgccgatcct gatcggcttt accgccgccc gcgaatttgg cggtaacccc tatctgggcg 2160 cgaccctcgg cgggatcctc acccacccgg cgctgaccaa cgcctggggc gtcgccgccg 2220 gcttccacac catgaatttc ttcggcatcg aagtggcgat gatcggctac cagggcaccg 2280 tcttcccggt gctgctggcg gtgtggttta tgagcatggt cgagaagcgg ctgcgccgcg 2340 tgatccctga cgcgctggac ctgatcctca ctccgttcct gacggtgatt atctccggct 2400 ttatcgccct gctgctgatc ggcccggccg gtcgcgcgct cggcgacggc atttcgttta 2460 tcctcagcac gcttatcagc catgccggct ggctggcggg cctgctgttc ggcggcctct 2520 attcggtgat cgtcattacc ggtatccatc acagcttcca tgccatcgag gccggactgc 2580 tgggcaaccc atcgattggc gtcaacttcc tgctgccgat ctgggcgatg gccaacgtcg 2640 cccagggcgg cgcctgcttt gcggtgtggt ttaaaaccaa agatgccaaa ataaaagcca 2700 tcaccctgcc gtcggcgttt tcggcgatgc tggggatcac cgaggcggca atcttcggga 2760 ttaacctgcg ctttgtgaaa ccgtttatcg ccgcgctggt gggcggtgcc gccggcggcg 2820 cctgggtggt gtcgatgcac gtctacatga ccgcggtggg cctgacggcg atcccgggaa 2880 tggctatcgt gcaggccagc tcgctgctga actacattat cggaatggcg atcgccttcg 2940 ccgtggcctt cgcgctctct ctgacgctga aatacaaaac ggacgctgaa taatgtcatt 3000 accgtcacgt ctgcctgcga tcctgcaggc cgttatgcag ggccagccgc aggcgctggc 3060 cgacagccat tatccgcaat ggcatctggc gccggtcaac ggactgctga acgatcctaa 3120 cggcttttgc caggtcgccg ggcgttacca cctgttttat cagtggaacc cgctcgcctg 3180 cgaccatacc tataagtgct ggggacactg gagctctgcc gatctgctgc actggcggca 3240 cgaacctatc gccctgatgc cggatgaaga gtatgaccgc aacggctgct actctggcag 3300 cgcggtcgag ttcgagggtg ccctgactct gtgctacacc ggcaacgtga aattccccga 3360 cggcgggcgc accgcctggc aatgtctggc gaccgagaat gccgatggca ccttccgcaa 3420 gctggggccg gtgctgccgc tgccagaagg ctataccggc catgtgcgcg accctaaagt 3480 gtggcggcag gacgggcgct ggtacatggt tcttggggcg caggatgtgc aacagcgcgg 3540 caaagtgctg ctgtttaccg ccagcgacct gcgggagtgg cgcctggtgg gcgagatcgc 3600 cgggcacgac gtgaacggcc tggcgaacgc cggctacatg tgggagtgcc cggatctctt 3660 tccgctggcg gacacccacc tgctgatctg ctgcccgcag gggctggccc gcgaagcgca 3720 gcgctttctc aatacctatc cggcggtgtg gatggcaggc cgcttcgacg ccgaacgcgg 3780 gatcttcgac cacggcccgc tgcacgagct ggacagcgga tttgagttct acgcgccgca 3840 gaccatgcag gccgacgatg gccgccgcct gctggtcggc tggatgggcg tccccgacgg 3900 ggacgagatg catcagccca cccgcgcgca gggctggatc catcagatga cctgcgtgcg 3960 tgagctggag tggcaggctg gcactctgta tcagcgtccg ctgcgcgagc tggtcgccct 4020 gcgcggggaa gcccagggct ggtgcggaca gaccctgccc ctcgccccga tggagctggc 4080 ctttgacctt tcccccgaca gcacgctggg gctggacttt gccggcgccc tgcagctcac 4140 cgtcaatcgc gacggcctac gtctgtcgcg ccgcggcctg cagacggcag agatgcatca 4200 ccgctactgg cgcggcgagg cgcgacgcct gcggatcttt atcgaccgct ccagcgtgga 4260 gattttcatc aacgatggcg agggggtgat gagcagccgc ttctttccgg gctatccggg 4320 gcagctcatc ttcagcggtg cgacgccggt ggcattctgc cgctggctgc tgcggccatg 4380 catggtagaa taa 4393 <210> 15 <211> 4423 <212> DNA <213> Salmonella typhimurium <400> 15 atgtacagaa aaagcacact tgcgatgctt atcgctttgc taaccagcgc tgcctcagcc 60 catgcgcaaa cggatataag caccattgaa gcccgactca acgcgctgga aaaacgcctg 120 caggaggcag aaaacagggc gcaaacggcg gaaaaccgcg ccggggcggc ggagaaaaaa 180 gttcagcaac tcaccgcgca gcagcaaaaa aaccagaact cgactcagga agtggctcag 240 cgtaccgcca gacttgagaa aaaagccgat gacaaaagcg gatttgagtt tcacggttac 300 gcccgctccg gcgtgataat gaatgattcc ggcgccagca ccaaatccgg agcctacata 360 acgccggcag gtgaaaccgg cggagctatc ggccgtctgg gaaaccaggc cgatacctat 420 gttgaaatga atcttgaaca taagcagacc ctggataatg gggccacgac ccgctttaag 480 gtgatggtcg ccgacgggca aacctcttat aacgactgga ctgcaagcac cagcgatctg 540 aacgttcgtc aggcctttgt cgaattgggt aacctgccga cgttcgctgg gccatttaag 600 ggctccaccc tgtgggccgg gaaacgtttc gaccgcgaca atttcgatat tcactggatt 660 gactctgatg tcgtgttcct cgccggtacc ggtggtggta tctatgacgt gaagtggaac 720 gacggcctgc ggagtaattt ctccctgtac gggcgtaact tcggcgacat tgatgattcc 780 agcaacagcg tgcagaacta tatcctcacc atgaatcact tcgcaggtcc gctgcagatg 840 atggtcagcg gtctgcgggc gaaggataac gacgagcgta aagatagcaa cggcaatctg 900 gcaaaaggcg atgcggcaaa caccggcgtg catgcgctgc tcggcctgca taacgacagt 960 ttctacggcc tgcgcgacgg tagcagtaaa accgctctgc tttatggtca tggtctgggc 1020 gcagaggtta aaggtatcgg atctgatggc gcacttcgtc cgggagccga cacatggcgc 1080 attgccagtt acggcaccac gccgctcagc gaaaactggt ctgttgcccc ggcaatgctg 1140 gcgcaacgca gtaaagaccg ctatgccgat ggcgacagct atcagtgggc aacattcaac 1200 ctgcgtctga ttcaggcaat caatcagaat ttcgctctcg cctacgaagg cagctaccag 1260 tacatggatc ttaaacccga aggttataac gatcgtcagg cggtgaacgg tagcttctac 1320 aagctcacct tcgccccgac atttaaggtc ggcagtatcg gtgatttctt cagtcgcccg 1380 gagattcgtt tctatacctc ctggatggac tggagcaaaa aactgaataa ttacgccagc 1440 gacgacgccc tgggcagtga cggttttaac tcgggcggcg aatggtcttt cggtgtgcag 1500 atggaaacct ggttctgacg cttacgcctg atgacaggaa tagccggggg tcagagcatc 1560 tttgtcaccc cggactcaac taagacgcag aaaaagcgct cccgtgaacg cgggacgaca 1620 acataaaaat gtttaagcct taagagggta ctatggattt tgaacagatt tcctgctcgc 1680 tgcttccgct tcttggaggc aaagaaaata tcgccagcgc cgcgcactgc gccacgcgcc 1740 tgcgcctggt gctggtcgat gattcgctgg ccgaccagca ggccatcggc aaagttgaag 1800 gggtgaaggg ctgttttcgt aatgccggac agatgcagat tattttcggc accggggtgg 1860 taaataaggt ctacgctgcc tttactcagg cggcgggtat tagcgaatcc agcaaatcgg 1920 aagccgccga catcgcggca aaaaagctca atccgttcca gcgcatcgcc cgcctgctat 1980 caaacatctt cgtgccgata atccctgcca tcgtcgcctc tggtctgctg atgggcctgc 2040 tgggaatggt caaaacatac ggctgggttg acccgggcaa cgccatctac atcatgctgg 2100 atatgtgcag ctcggcggca tttatcattc tgccgattct gattggcttt accgccgccc 2160 gcgaattcgg cggtaatcct tatctcggcg cgacgcttgg cggcattctg actcatccag 2220 cgctgactaa cgcctggggc gtggccgcgg gtttccacac catgaacttt ttcggcttcg 2280 aaattgccat gatcggctat cagggtacgg tgttcccggt actgctggca gtatggttta 2340 tgagcatcgt tgagaagcag ttgcgtcgcg caatccccga tgccctggat ttgatcctga 2400 cgccgttcct gacggtgatt atatccggtt ttatcgccct gttgattatc ggcccggccg 2460 gtcgcgcact gggcgacggt atctcgtttg tcctcagcac cctgattagc cacgccggct 2520 ggctcgccgg gttactgttt ggcggtctct attcagttat cgtcattacc ggtattcatc 2580 acagcttcca tgcggttgaa gccgggttgc tgggcaatcc ctccatcggc gtcaacttcc 2640 tgctgccgat ttgggcgatg gccaacgtcg ctcagggcgg agcctgtctg gcggtgtggt 2700 tcaaaaccaa agatgcaaaa attaaagcca ttactctgcc ctcggcgttt tccgccatgc 2760 tgggcatcac cgaggcggcg atttttggta ttaacctgcg ctttgtgaag ccatttattg 2820 cggcgctgat tggtggtgcg gcgggcggcg catgggtggt atctgtacac gtctacatga 2880 ccgcggtcgg cttgacagcg atccccggca tggccatcgt gcaggccagt tcgctgttga 2940 actacattat cgggatggtt atcgcctttg gcgtcgcctt tacggtctcc ctggttttga 3000 aatacaaaac ggacgctgaa taatgtctct tccatcacga ctgcctgcga ttttgcaggc 3060 cgtaatgcag ggccagccgc gcgcgctggc cgatagccac tatccgcgct ggcaccatgc 3120 gccggtcacc gggctgatga acgaccccaa cggctttatc gaatttgccg gacgctatca 3180 tctgttttat cagtggaacc cgctcgcctg cgatcatacg tttaagtgct gggcgcactg 3240 gagttccatc gatctgctgc actggcagca tgagcccatt gcgctgatgc cggacgaaga 3300 gtatgaccgt aacggctgct actccggcag cgcggtggat aacaacggta cgcttaccct 3360 gtgctatacc ggcaacgtga agtttgccga gggagggcga accgcctggc aatgcctggc 3420 aacggaaaac gctgacggca ccttccgcaa aatcggtccg gtcctgccgc tgccggaggg 3480 ctacaccggc cacgtgcgcg acccaaaagt ctggcgacac gaagacctgt ggtacatggt 3540 gctgggcgcg caggatcggc aaaagcgcgg caaggtgctg ctgttcagct ctgcggatct 3600 ccatcagtgg acgagtatgg gtgaaatcgc cggccacggc atcaatggcc tcgacgacgt 3660 cggctatatg tgggagtgcc cggatctttt tccactcggc gaccagcata ttctaatctg 3720 ctgtccgcag gggattgccc gtgaggaaga gtgctacctg aacacctacc cggcagtatg 3780 gatggcgggc gagtttgatt acgctgctgg cgctttcaga cacggcgaac tgcacgaact 3840 ggacgccggg tttgagttct acgccccgca aaccatgctt accagtgatg gccgtcgtct 3900 gctggtcggc tggatgggcg tgccggaggg cgaagagatg cttcagccga ccctgaacaa 3960 cggctggatc catcagatga cctgcctgcg tgagctggag tttatcaacg gtcagctcta 4020 tcagcgtccg ctacgggaac tgagcgccct gcgcggtgaa gcgaacggct ggtcggggaa 4080 cgccctgccg ctggcaccga tggaaatcga tttgcaaacc cgcgggggcg atatgttgag 4140 cctcgatttt ggcggcgtat taacccttga gtgcgatgcc agcggactcc gcctggcccg 4200 acgcagtctc gccagtgacg agatgcatta tcgttactgg cgcggaaacg tccgctcgct 4260 gcgtgttttc atcgaccagt cgagcgtgga gattttcata aacggcggtg aaggggtgat 4320 gagcagccgc tacttcccgg cctgctccgg tcagctaaca ttctccggca tcacgccgga 4380 cgcattctgc tactggccgc tgcgaacttg catggtagaa taa 4423 <210> 16 <211> 3883 <212> DNA <213> Eastern equine encephalomyelitis virus (Eastern equine encephalomyelitis virus) <400> 16 ctatattgct gaaggtacag gcgtttccat aactatttgc tcgcgttttt tactcaagaa 60 gaaaatgcca atagcaaca tcaggcagac aatacccgaa attgcgaaga aaactgtctg 120 gtagcctgcg tggtcaaaga gtatcccagt cggcgttgaa agcagcacaa tcccaagcga 180 actggcaatt tgaaaaccaa tcagaaagat cgtcgacgac aggcgcttat caaagttttgc 240 cacgctgtat ttgaagacgg atatgacaca aagtggaacc tcaatggcat gtaacaactt 300 cactaatgaa ataatccagg ggttaacgaa cagcgcgcag gaaaggatac gcaacgccat 360 aatcacaact ccgataagta atgcattttt tggccctacc cgattcacaa agaaaggaat 420 aatcgccatg cacagcgctt cgagtaccac ctggaatgag ttgagataac catacaggcg 480 cgttcctaca tcgtgtgatt cgaataaacc tgaataaaag acaggaaaaa gttgttgatc 540 aaaaatgtta tagaaagacc acgtccccac aataaatatg acgaaaaccc agaagtttcg 600 atccttgaaa actgcgataa aatcctcttt ttttacccct cccgcatctg ccgctacgca 660 ctggtgatcc ttatctttaa aacgcatgtt gatcatcata aatacagcgc caaatagcga 720 gaccaaccag aagttgatat ggggactgat actaaaaaaat atgccggcaa agaacgcgcc 780 aatagcatag ccaaaagatc cccaggcgcg cgctgttcca tattcgaaat gaaaatttcg 840 cgccattttt tcggtgaagc tatcaagcaa accgcatccc gccagatacc ccaagccaaa 900 aaatagcgcc cccagaatta gacctacaga aaaattgctt tgcagtaacg gttcataaac 960 gtaaatcata aacggtccgg tcaagaccag gatgaaactc atacaccaga tgagcggttt 1020 cttcagaccg agtttatcct gaacgatgcc gtagaacatc ataaatagaa tgctggtaaa 1080 ctggttgacc gaataaagtg tacctaattc cgtccctgtc aaccctagat gtcctttcag 1140 ccaaatagcg tataacgacc accacagcga ccaggaaata aaaaagagaa atgagtaact 1200 ggatgcaaaa cgatagtacg catttctgaa tggaatattc agtgccataa ttacctgcct 1260 gtcgttaaaa aattcacgtc ctatttagag ataagagcga cttcgccgtt tacttctcac 1320 tattccagtt cttgtcgaca tggcagcgct gtcattgccc ctttcgccgt tactgcaagc 1380 gctccgcaac gttgagcgag atcgataatt cgtcgcattt ctctctcatc tgtagataat 1440 cccgtagagg acagacctgt gagtaacccg gcaacgaacg catctcccgc ccccgtgcta 1500 tcgacacaat tcacagacat tccagcaaaa tggtgaactt gtcctcgata acagaccacc 1560 accccttctg cacctttagt caccaacagc atggcgatct catactcttt tgccagggcg 1620 catatatcct gatcgttctg tgtttttcca ctgataagtc gccattcttc ttccgagagc 1680 ttgacgacat ccgccagttg tagcgcctgc cgcaaacaca agcggagcaa atgctcgtct 1740 tgccatagat cttcacgaat attaggatcg aagctgacaa aacctccggc atgccggatc 1800 gccgtcatcg cagtaaatgc gctggtacgc gaaggctcgg cagacaacgc aattgaacag 1860 agatgtaacc attcgccatg tcgccagcag ggcaagtctg tcgtctctaa aaaaagatcg 1920 1980 accgtggatg tccggtgcca ttcatcttgc ttcagatacg tgatatcgac tccctcagtt 2040 agcagcgttc tttgcattaa cgcaccaaaa ggatcatccc ccacccgacc tataaaccca 2100 cttgttccgc ctaatctggc gattcccacc gcaacgttag ctggcgcgcc gccaggacaa 2160 ggcagtaggc gcccgtctga ttctggcaag agatctacga ccgcatcccc taaaacccat 2220 actttggctg acattttttt cccttaaatt catctgagtt acgcatagtg ataaacctct 2280 ttttcgcaaa atcgtcatgg atttactaaa acatgcatat tcgatcacaa aacgtcatag 2340 ttaacgttaa catttgtgat attcatcgca tttatgaaag taagggactt tatttttata 2400 aaagttaacg ttaacaattc accaaatttg cttaaccagg atgattaaaa tgacgcaatc 2460 tcgattgcat gcggcgcaaa acgccctagc aaaacttcat gagcaccggg gtaacacttt 2520 ctatccccat tttcacctcg cgcctcctgc cgggtggatg aacgatccaa acggcctgat 2580 ctggtttaac gatcgttatc acgcgtttta tcaacatcat ccgatgagcg aacactgggg 2640 gccaatgcac tggggacatg ccaccagcga cgatatgatc cactggcagc atgagcctat 2700 tgcgctagcg ccaggagacg ataatgacaa agacgggtgt ttttcaggta gtgctgtcga 2760 tgacaatggt gtcctctcac ttatctacac cggacacgtc tggctcgatg gtgcaggtaa 2820 tgacgatgca attcgcgaag tacaatgtct ggctaccagt cgggatggta ttcatttcga 2880 gaaacagggt gtgatcctca ctccaccaga aggaatcatg cacttccgcg atcctaaagt 2940 gtggcgtgaa gccgacacat ggtggatggt agtcggggcg aaagatccag gcaacacggg 3000 gcagatcctg ctttatcgcg gcagttcgtt gcgtgaatgg accttcgatc gcgtactggc 3060 ccacgctgat gcgggtgaaa gctatatgtg ggaatgtccg gactttttca gccttggcga 3120 tcagcattat ctgatgtttt ccccgcaggg aatgaatgcc gagggataca gttaccgaaa 3180 tcgctttcaa agtggcgtaa tacccggaat gtggtcgcca ggacgacttt ttgcacaatc 3240 cgggcatttt actgaacttg ataacgggca tgacttttat gcaccacaaa gctttttagc 3300 gaaggatggt cggcgtattg ttatcggctg gatggatatg tgggaatcgc caatgccctc 3360 aaaacgtgaa ggatgggcag gctgcatgac gctggcgcgc gagctatcag agagcaatgg 3420 caaacttcta caacgcccgg tacacgaagc tgagtcgtta cgccagcagc atcaatctgt 3480 ctctccccgc acaatcagca ataaatatgt tttgcaggaa aacgcgcaag cagttgagat 3540 tcagttgcag tgggcgctga agaacagtga tgccgaacat tacggattac agctcggcac 3600 tggaatgcgg ctgtatattg ataaccaatc tgagcgactt gttttgtggc ggtattaccc 3660 acacgagaat ttagacggct accgtagtat tcccctcccg cagcgtgaca cgctcgccct 3720 aaggatattt atcgatacat catccgtgga agtatttatt aacgacgggg aagcggtgat 3780 gagtagtcga atctatccgc agccagaaga acgggaactg tcgctttatg cctcccacgg 3840 agtggctgtg ctgcaacatg gagcactctg gctactgggt taa 3883

Claims

1. A genetically engineered microbial cell for producing a carbohydrate of interest, wherein due to reduced and / or decreased expression and / or activity of at least one protein that leads to consumption of intracellular phosphosugars within the genetically engineered microbial cell, the microbial cell has increased intracellular availability of at least one phosphosugar compared to a wild-type cell, the microbial cell is an Escherichia coli strain that displays the genotype lacY + , lacZ - , fucI K - , wcaJ - , pfkA - , and overexpresses the enzymes ManB, ManC, Gmd, and WcaG for de novo synthesis of GDP-fucose, the 2’-fucosyltransferase gene wbgL from E. coli: 0126, the sugar efflux transporter yberc0001_9420 from Yersinia pseudotuberculosis ATCC 43970, and the csc gene cluster of E. coli W, wherein the accession number for E. coli W is CP002185.1, the csc gene cluster comprises genes for sucrose permease, fructokinase, sucrose hydrolase, and the transcriptional repressor genes cscB, cscK, cscA, and cscR, and produces a carbohydrate of interest when cultured in a medium comprising a mixed monosaccharide feedstock as a carbon source and energy source, and with lactose added as an acceptor substrate, wherein the mixed monosaccharide feedstock consists of glucose and fructose, and the carbohydrate of interest is 2’-fucosyllactose.

2. The genetically engineered microbial cell of claim 1, wherein the genetically engineered microbial cell further comprises a UDP-galactose biosynthetic pathway for intracellular formation of UDP-galactose.

3. The genetically engineered microbial cell of claim 1 or 2, wherein the genetically engineered microbial cell further comprises a GDP-fucose biosynthetic pathway for intracellular formation of GDP-L-fucose.

4. The genetically engineered microbial cell of claim 1 or 2, wherein the genetically engineered microbial cell further comprises a UDP-N-acetylglucosamine biosynthetic pathway for intracellular formation of UDP-N-acetylglucosamine.

5. The genetically engineered microbial cell of claim 1 or 2, wherein the genetically engineered microbial cell further comprises at least one glycosyltransferase.

6. The genetically engineered microbial cell of claim 1 or 2, wherein the genetically engineered microbial cell further comprises enhanced synthesis of phosphoenolpyruvate.

7. The genetically engineered microbial cell of claim 1 or 2, wherein the genetically engineered microbial cell further comprises at least one monosaccharide transporter for transporting monosaccharides from the culture medium into the cytoplasm of the microbial cell.

8. The genetically engineered microbial cell of claim 1 or 2, wherein the genetically engineered microbial cell further comprises at least one exporter protein or permease that exports a carbohydrate of interest from the cell.

9. Use of the genetically engineered microbial cell of any one of claims 1 to 8 for producing 2’ fucosyllactose.

10. A method for fermentative production of a carbohydrate of interest, the method comprising: a) providing a genetically engineered microbial cell capable of producing a carbohydrate of interest, wherein due to reduced and / or decreased expression and / or activity of at least one protein leading to consumption of intracellular phosphosugars within said genetically engineered microbial cell, said microbial cell has increased intracellular availability of at least one phosphosugar compared to a wild type cell, said microbial cell is showing the genotype lacY + , lacZ - , fuclK - , wcaJ - , pfkA - , and overexpressing the enzymes ManB, ManC, Gmd and WcaG for de novo synthesis of GDP-fucose, the 2' fucosyltransferase gene wbgL from Escherichia coli: 0126, the sugar efflux transporter yberc0001_9420 from Yersinia pseudotuberculosis ATCC 43970, and the csc gene cluster of Escherichia coli strain W, wherein said Escherichia coli W has the accession number CP002185.1, said csc gene cluster comprises genes for sucrose permease, fructokinase, sucrose hydrolase, and the transcriptional repressor genes cscB, cscK, cscA and cscR; b) culturing the genetically engineered microbial cell in a culture medium that allows production of the carbohydrate of interest, wherein the culture medium comprises a mixed monosaccharide feedstock as carbon source and energy source, wherein the mixed monosaccharide feedstock consists of glucose and fructose, and lactose is added as acceptor substrate; and c) recovering the carbohydrate of interest; wherein the carbohydrate of interest is 2’ -fucosyllactose.

Images