Reduction of pantothenic acid levels during fermentative production of oligosaccharides.

Abstract

Disclosed are means and methods for the fermentative production of oligosaccharides of interest by genetically engineered microbial cells, in which the concomitant biosynthesis of pantothenic acid is reduced or abolished.

Description

[Technical field]

[0001] The present invention relates to the fermentative production of oligosaccharides of interest and to genetically engineered microbial cells for the production of oligosaccharides of interest, more particularly, the present invention relates to the fermentative production of oligosaccharides of interest, which are suitable for use in infant formulas. [Background technology]

[0002] Breastfeeding, also called lactation, is the method of feeding a baby with a mother's breast milk, whether the milk is given directly from the breast or collected from the breast and then fed to the baby in a bottle. Breastfeeding is widely advocated as the "ideal" for infants because of the health benefits of breast milk. Health organizations, including the World Health Organization (WHO), recommend exclusive breastfeeding for at least the first six months of an infant's life. Despite this recommendation, fewer than 40% of the world's infants are exclusively breastfed under the age of six months. Instead, infants are fed manufactured foods called infant formulas (IF), which are usually prepared for artificial feeding from powders or ready-to-use liquid preparations. The United States Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food that claims or is represented for special dietary use solely as an infant food because of its suitability as a simulant of human milk or as a total or partial substitute for human milk."

[0003] In 2001, a WHO report found that infant formulas prepared according to the appropriate Codex Alimentarius standards are safe complementary foods and suitable breast-milk substitutes. In 2003, WHO and the United Nations Children's Fund (UNICEF) published a Global Strategy on Infant Feeding, which reiterated that "processed foods for young children, when sold or otherwise distributed, shall meet the applicable standards recommended by the Codex Alimentarius Commission" and also warned that "lack of breast-feeding, especially exclusive breast-feeding, during the first six months of life is an important risk factor for infant morbidity and mortality." In this sense, infant formula is one of the most regulated foods in the world.

[0004] The nutritional value of infant formulas and the content of ingredients including nutrients, vitamins and minerals are strictly regulated. For example, the nutritional content of infant formulas sold in the United States is regulated by the Food and Drug Administration (FDA) based on the recommendations of the American Academy of Pediatrics Committee on Nutrition. The addition of prebiotic oligosaccharides such as galactooligosaccharides (GOS), fructooligosaccharides (FOS) and human milk oligosaccharides (HMO) to infant formulas has been permitted for several years.

[0005] Prebiotic oligosaccharides are non-digestible carbohydrates, i.e. carbohydrates that humans cannot metabolize. However, prebiotic carbohydrates can be metabolized by certain non-pathogenic gut bacteria. Promoting the growth of these gut bacteria by supplying prebiotic oligosaccharides helps prevent bacterial infections by outcompeting pathogens for nutrients and by impairing growth through the secretion of acidic metabolic products by non-pathogenic bacteria, thereby lowering the pH in the gut. Therefore, prebiotics are added to infant formulas to promote the development of a "healthy" gut microbiota in infants.

[0006] GOS and FOS are not naturally occurring prebiotic carbohydrates and represent a family of oligosaccharides with limited complexity. However, a much more diverse and complex family of prebiotic oligosaccharides is found in human milk. This family of oligosaccharides is called human milk oligosaccharides (HMO), and the family of HMOs appears to be more advantageous for healthy infants than artificial surrogates.

[0007] HMOs are found exclusively in human milk and represent the third most abundant solid content in human milk. HMOs have a prebiotic effect and thus shape the gut microbiota by promoting the growth of beneficial bacteria, such as Bifidobacteria and Lactobacilli, suppressing the growth of pathogenic bacteria, inhibiting the binding of bacterial and viral pathogens to epithelial cells, supporting the development of the infant's immune system, and enhancing brain development and cognitive abilities. In addition to their beneficial effects on infant development, HMOs are also thought to improve the gut microbiota and adult health.

[0008] More than 150 structurally distinct HMOs have been identified so far. They are all composed of five or fewer different monosaccharide moieties, namely glucose, galactose, L-fucose, N-acetylglucosamine and N-acetylneuraminic acid (sialic acid). Based on their chemical composition, HMOs are divided into neutral and acidic HMOs. Members of the latter subset of HMOs contain at least one sialic acid (i.e., N-acetylneuraminic acid) moiety and are therefore also called "sialylated HMOs". Neutral HMOs may or may not contain a fucose moiety.

[0009] Milk from some dairy cows contains oligosaccharides with structural homology to HMOs, but the oligosaccharide composition of cow's milk is less complex than that of human milk, and the amount of oligosaccharides in cow's milk is much lower than that in human milk.

[0010] It does not appear feasible to isolate large quantities of specific oligosaccharides from milk, particularly from milk of dairy cows. Oligosaccharides can be synthesized chemically or by in vitro biocatalysis. However, chemical synthesis of oligosaccharides involves the stepwise addition of monosaccharide moieties to the reducing or non-reducing ends of existing glycans, which addition involves adding and removing protecting groups to and from side chains within the monosaccharide moieties of the glycans. Given the stereochemical specificity, chemical synthesis has also proven not feasible to provide oligosaccharides at reasonable cost on an industrial scale and in sufficiently sustainable conditions to be used in the food industry. Instead, oligosaccharides can be produced by in vitro biochemical reactions, i.e., biocatalysis, where enzymes catalyze the formation of glycosidic bonds between sugar moieties. However, biocatalysis is also not feasible at reasonable cost for industrial scale production of oligosaccharides due to the expensive educts required, e.g., nucleotide-activated monosaccharides or complex oligosaccharides.

[0011] To overcome the above-outlined shortcomings in the production of HMOs, and to provide HMOs on an industrial scale at a reasonable cost so that they can be reasonably priced for use in infant formulas, fermentation production methods using metabolically engineered bacterial cells, such as Escherichia coli, have been developed. Such in vivo production approaches overcome the need to use expensive educts, since the metabolically engineered bacteria can synthesize the educts and the desired HMOs intracellularly. The desired HMOs can then be purified from the bacteria or the fermentation broth. Such in vivo production methods have been proven to provide HMOs in a sufficiently pure form and at an economically viable cost for use as an ingredient in infant formulas.

[0012] Referring to the prescribed nutritional content of infant formulas sold in the United States previously described herein, the following compounds must be included in all infant formulas produced in the United States: protein, fat, linoleic acid, vitamins A, C, D, E, K, thiamine, riboflavin, vitamins B6 and B 12, niacin, folic acid, pantothenic acid, calcium, magnesium, iron, zinc, manganese, copper, phosphorus, iodine, sodium chloride, potassium chloride, carbohydrates as well as nucleotides. In this regard, the US FDA has issued nutritional specifications in regulation 21 CRF 107.100, prescribing a minimum concentration of pantothenic acid of at least 0.3 mg / 100 kcal. Infant formulas currently contain pantothenic acid in concentrations of about 0.5 to about 0.9 mg / 100 kcal.

[0013] According to EU Regulation (EU) No. 609 / 2013 of 25 September 2015 and its complementary Commission Regulation (EU) 2016 / 127, the concentration of pantothenic acid in infant formulae must be not less than 0.4 mg / 100 kcal and must not exceed 2 mg / 100 kcal when prepared for consumption as indicated by the manufacturer.

[0014] Pantothenate (vitamin B5), a salt of pantothenic acid (both terms are used interchangeably in this disclosure unless otherwise specified), is an important precursor for the biosynthesis of coenzyme A (CoA) and carrier proteins with phosphopantetheine prosthetic groups. Coenzyme A is an essential cofactor for cell growth and is involved in many metabolic reactions, including the synthesis of phospholipids, the synthesis and degradation of fatty acids, and the operation of the tricarboxylic acid cycle.

[0015] Most prokaryotes, fungi, and plants can synthesize pantothenate de novo, whereas animals rely on exogenous vitamin B5 supplies. In bacteria such as Escherichia coli (E. coli), the biosynthesis of pantothenic acid involves several steps (Figure 1). α-ketoiso-valerate is converted to α-ketopantoate by ketopantoate hydroxymethyltransferase (PanB; UniProtKB accession no. P31057 for E. coli K12 PanB). α-ketopantoate is then reduced to pantoate by ketopantoate reductase PanE (UniProtKB accession no. P0A9J4 for E. coli K12 PanE). Pantothenate synthetase (PanC; UniProtKB accession no. P31663 for E. coli K12 PanC) catalyzes the condensation of pantoate and β-alanine to pantothenate. β-alanine is produced from aspartate by aspartate α-decarboxylase (PanD; UniProtKB accession no. P0A790 for E. coli K12 PanD). Genes encoding PanB and PanC form an operon panBC in E. coli, whereas the panD gene is located downstream of the operon separated by only one gene. PanE, encoding the NADPH-dependent keto-pantoate reductase, is located in another part of the E. coli genome and forms an operon with yajL, which is not involved in pantothenate synthesis.

[0016] Pantothenate is further converted to coenzyme A by several enzymatic reactions. However, bacteria also secrete pantothenate into their environment, since the production of pantothenate in E. coli is several times higher than its intracellular demand and thus has been found to accumulate in the fermentation medium (Jackowski, S. and Rock, CO (1984) J. Bacteriol. 158, 115-120). E. coli can also internalize exogenous pantothenate, which is mediated by their pantothenate permease PanF (UniProtKB accession no. P16256 for E. coli K12 PanF), a sodium / pantothenate symporter.

[0017] Pantothenic acid is a by-product found in the fermentation broth when culturing Escherichia coli (E. coli) for HMO production. Given the prescribed maximum amount of vitamin B5 in infant formula, it may be necessary to deplete pantothenic acid from the HMO preparation to make the HMO product suitable as an ingredient in infant formula. This is particularly appropriate when the HMO concentration in the infant formula should be similar to the natural concentration of HMO in breast milk, i.e., about 40 g to about 120 g of HMO per kg of ready to use infant formula.

[0018] It may be possible to reduce pantothenic acid levels in process streams obtained from fermentation broths containing HMOs by crystallizing the HMOs from the process stream. The HMOs may be crystallized from water, organic solvents, or mixtures of water and organic solvents. The crystallization of HMOs may be improved by adding an organic acid to the HMO-containing solution.

[0019] Additionally and / or alternatively, pantothenic acid can be separated from the oligosaccharides of interest by subjecting the process stream to at least one specific ion exchange process step for the removal of pantothenic acid, i.e. anionic ion exchange chromatography or adsorption of pantothenic acid onto an anion adsorption resin. The anion exchange resin or anion adsorption resin may be a strong anion exchange resin or a weak anion exchange resin, preferably a weak anion exchange resin.

[0020] The approaches outlined above for removing pantothenic acid require additional processing steps in the purification scheme for producing HMOs by fermentation means. Furthermore, using such specific ion exchange processing steps to remove pantothenic acid from the process stream may be feasible for purifying neutral HMOs. However, such ion exchange processing steps cannot be used for the purification and recovery of acidic sialylated HMOs such as sialyllactose, because acidic HMOs have similar binding capacity to the anion exchange resins used as pantothenic acid. This makes it virtually impossible to separate sialylated HMOs from pantothenic acid on an industrial scale using ion exchange processing to remove pantothenic acid. Summary of the Invention [Problem to be solved by the invention]

[0021] To overcome the aforementioned drawbacks, methods have been developed for the fermentative production of oligosaccharides of interest, in which the accumulation of pantothenic acid in the culture medium during oligosaccharide biosynthesis by microbial cells is avoided or at least significantly reduced. [Means for solving the problem]

[0022] In a first aspect, a genetically engineered microbial cell for producing an oligosaccharide of interest is provided, the genetically engineered microbial cell having a metabolic pathway for intracellular biosynthesis of the oligosaccharide of interest and having low levels or no intracellular biosynthesis of pantothenic acid.

[0023] In a second aspect, there is provided the use of a genetically engineered microbial cell that has a metabolic pathway for the intracellular biosynthesis of an oligosaccharide of interest and has low levels or no intracellular biosynthesis of pantothenic acid to produce the oligosaccharide of interest.

[0024] In a third aspect, a method for producing an oligosaccharide of interest is provided, the method comprising (i) providing a genetically engineered microbial cell having a metabolic pathway for intracellular biosynthesis of the oligosaccharide of interest and having low levels of intracellular biosynthesis of pantothenic acid or no intracellular biosynthesis of pantothenic acid, (ii) culturing the genetically engineered microbial cell in a medium under conditions that permit the microbial cell to synthesize the oligosaccharide of interest intracellularly, and optionally (iii) recovering the oligosaccharide of interest.

[0025] In a fourth aspect, a preparation of an oligosaccharide of interest is provided, the preparation being obtained by a method in which genetically engineered microbial cells, having a metabolic pathway for intracellular biosynthesis of the oligosaccharide of interest and having low levels of intracellular biosynthesis of pantothenic acid or no intracellular biosynthesis of pantothenic acid, are cultured in a medium and under conditions that allow the microbial cells to synthesize the oligosaccharide of interest intracellularly, the preparation of oligosaccharide containing low levels of pantothenic acid or no pantothenic acid.

[0026] In a fifth aspect, there is provided the use of a preparation of an oligosaccharide of interest for the manufacture of a nutritional composition.

[0027] In a sixth aspect, a nutritional composition is provided that contains a preparation of an oligosaccharide of interest, the preparation containing low levels of pantothenic acid or no pantothenic acid. [Brief description of the drawings]

[0028] [Figure 1]FIG. 1 is a diagram showing the metabolic pathway for the intracellular biosynthesis of pantothenic acid in E. coli as shown in Leonardi, R. and Jackowski, S. "Biosynthesis of Pantothenic Acid and Coenzyme A." EcoSal Plus. 2007 Apr;2(2):10.1128 / ecosalplus.3.6.3.4. [Figure 2-1] FIG. 2 is a diagram showing the amino acid sequence of Escherichia coli K-12 ketopantoate reductase PanE (UniProtKB Entry number P0A9J4, UniProt release 2021_03 as published June 02, 2021). [Figure 2-2] FIG. 2 is a diagram showing the amino acid sequence of Escherichia coli K-12 ketopantoate reductase PanE (UniProtKB Entry number P0A9J4, UniProt release 2021_03 as published June 02, 2021). [Diagram 3] Figure 3 shows two graphs showing the growth of two different E. coli strains that produce 3'-sialyllactose and the amount of pantothenic acid in their medium, where Figure 3A shows the growth and pantothenic acid levels in the culture medium of the precursor strain before it was engineered to reduce pantothenic acid biosynthesis, and Figure 3B shows the growth and pantothenic acid levels in the culture medium of an E. coli strain engineered to have low levels of intracellular pantothenic acid biosynthesis. [Figure 4]Figure 4 is two graphs showing the growth of two different lacto-N-tetraose producing E. coli strains and the amount of pantothenic acid in their culture medium: Figure 4A shows the growth and pantothenic acid levels in the culture medium of the precursor strain before it was engineered to reduce pantothenic acid biosynthesis, and Figure 4B shows the growth and pantothenic acid levels in the culture medium of an E. coli strain engineered to have low levels of intracellular pantothenic acid biosynthesis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] According to a first aspect, there is provided a genetically engineered microbial cell for producing an oligosaccharide of interest, the microbial cell having a metabolic pathway for the intracellular biosynthesis of the oligosaccharide of interest, the genetically engineered microbial cell also having low levels of intracellular biosynthesis of pantothenic acid or no intracellular biosynthesis of pantothenic acid.

[0030] A genetically engineered microbial cell is a microbial cell that has been genetically engineered to have a metabolic pathway for the intracellular biosynthesis of an oligosaccharide of interest and / or to have low levels of pantothenic acid biosynthesis or to be incapable of pantothenic acid biosynthesis.

[0031] The term "microbial cell" as used herein refers to a unicellular organism. A microbial cell may be a prokaryotic or eukaryotic cell. Suitable prokaryotic cells include bacterial and archaeal cells. Suitable eukaryotic cells include yeast and fungal cells.

[0032] In further and / or alternative embodiments, the prokaryotic cell is a bacterial cell, preferably a bacterium of a genus selected from the group consisting of Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Micromonospora, Pseudomonas, Rhodococcus, and Sporolactobacillus. Suitable bacterial species within these genera include Bacillus subtilis, B. licheniformis, B. coagulans, B. thermophilus, B. laterosporus, B. mega-terium, B. mycoides, B. pumilus, B. lentus, B. cereus, B. circulans, Bifido-bacterium longum, B. infantis, B. bifidum, Citrobacter freundii, and B. erythrocytes. freundii, Clostridium cellulolyticum, C. ljungdahlii, C. autoethanogenum, C. acetobutylicum, Corynebacterium glutamicum, E. enterococcus faecium, E. thermophiles,thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acidophilus acidophilus, L. salivarius, L. plantarum, L. helveticus, L. delbrueckii, L. rhamnosus, L. bulgaricus, L. crispatus, L. gasseri, L. casei, L. reuteri, L. jensenii, L. lactis, Pantoea citrea, Pectobacterium carotovorum, Propionibacterium freudenreichii freudenreichii, Pseudomonas fluorescens, P. aeruginosa, Streptococcus thermophiles and Xanthomonas campestris.

[0033] In some embodiments, the eukaryotic cell is a yeast cell, preferably a yeast cell selected from the genera of the group consisting of Saccharomyces sp., Saccharomycopsis sp., Pichia sp., Hansenula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp., and Schizosaccharomyces sp. In further and / or alternative embodiments, the yeast cell is a Saccharomyces cerevisiae cell, a Pichia pastoris cell, or a Hanensula polymorpha cell.

[0034] The genetically engineered microbial cell is a microbial cell for producing a desired oligosaccharide.

[0035] As used herein, the term "oligosaccharide" refers to a polymeric sugar molecule that typically contains at least three monosaccharide moieties, but not more than 12 monosaccharide moieties, preferably not more than 10 monosaccharide moieties. The monosaccharide moieties of an oligosaccharide are linked to each other by glycosidic bonds. An oligosaccharide may consist of a linear chain of monosaccharide moieties, or an oligosaccharide may comprise a branched chain of monosaccharide moieties, at least one monosaccharide moiety having at least three monosaccharide moieties attached to it by glycosidic bonds. The monosaccharide portion of the oligosaccharide may be selected from the group consisting of aldoses (e.g., arabinose, xylose, ribose, desoxyribose, lyxose, glucose, idose, galactose, talose, allose, altrose, mannose), ketoses (e.g., ribulose, xylulose, fructose, sorbose, tagatose), deoxysugars (e.g., rhamnose, fucose, quinovose), deoxy-aminosugars (e.g., N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine), uronic acids (e.g., galacturons, glucurons), and ketoaldonic acids (e.g., N-acetyl-neuraminic acid).

[0036] The expression "oligosaccharide of interest" as used herein refers to an oligosaccharide that is believed to be synthesized by a microbial cell. Typically, the microbial cell has been selected and / or genetically engineered to synthesize a specific oligosaccharide, i.e., the oligosaccharide of interest. Any other oligosaccharide that may be synthesized by a microbial cell other than the oligosaccharide of interest is considered to be a by-product, regardless of whether the other oligosaccharide other than the oligosaccharide of interest is a reaction product of an intermediate reaction in the biosynthesis of the oligosaccharide of interest or constitutes the end product of another metabolic pathway other than the metabolic pathway for the biosynthesis of the oligosaccharide of interest.

[0037] In some embodiments, the oligosaccharide of interest is a human milk oligosaccharide, i.e. an oligosaccharide selected from the group of oligosaccharides present in human milk (HMO). Human milk oligosaccharides constitute a diverse mixture of indigestible oligosaccharides present in human milk. Human milk is unique with respect to its oligosaccharide composition and amount. More than 150 structurally different HMOs have been identified so far. Most HMOs are characterized by a lactose moiety (Gal-β1,4-Glc) at their reducing end. Many HMOs contain a fucose moiety and / or a sialic acid moiety. More generally, the monosaccharides from which HMOs are derived are D-glucose, D-galactose, N-acetyl-glucosamine, L-fucose and / or N-acetylneuraminic acid.

[0038] In further and / or alternative embodiments, the oligosaccharide of interest is 2'-fucosyl-lactose (2'-FL), 3-fucosyl-lactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-triose II, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucco-pentaose I (LNnFP-I), lacto-N-fucopentaose II (LNFP ... II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofuc-pentaose V (LNnFP-V), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto-N-difucosyl ... Fucosyl-lacto-N-sialylpentaose I, lacto-N-difucosyl-hexaose II, para-lacto-N-fucosyl-hexaose (paraLNH), fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyl-lactose (3F-3'- SL), 3-fucosyl-6'-sialyllactose (3F-6'-SL), lacto-N-neodifuco-hexaose I, 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyllactose-N-tetraose a (LST-a), sialyllactose-N-tetraose b (LST-b), sialyllactose-N-tetraose c (LST-c), disialyl-lacto-N-tetraose (DS-LNT), disialyl-lacto-N-fuco-pentaose (DS-LNFP V), lacto-N-neodifuco-hexaose I (LNnDFH I), 3'-galactosyllactose (3'-GL), 6'-galactosyllactose (6'-GL).

[0039] The genetically engineered microbial cells are genetically modified for the production of an oligosaccharide of interest. Typically, the oligosaccharide of interest is an oligosaccharide that is not naturally synthesized by the microbial cells. Thus, the microbial cells are usually genetically engineered to be capable of synthesizing the oligosaccharide of interest intracellularly, i.e., the microbial cells are genetically engineered to synthesize the oligosaccharide of interest intracellularly when cultured in a medium under conditions that allow the synthesis of the oligosaccharide of interest. Thus, the genetically engineered microbial cells are genetically engineered to have a metabolic pathway for the intracellular biosynthesis of the oligosaccharide of interest.

[0040] The engineered microbial cell has a metabolic pathway for the intracellular biosynthesis of a desired oligosaccharide, and thus has all the enzymes and transporters necessary to provide an acceptor sugar from which to obtain a monosaccharide moiety, provide a donor substrate for the monosaccharide moiety to be transferred to the acceptor sugar, and catalyze the conversion of the acceptor sugar to the desired oligosaccharide by transferring the monosaccharide moiety from the donor substrate to the acceptor sugar.

[0041] In some embodiments, the acceptor sugar is a disaccharide that is converted to a trisaccharide. In other embodiments, the acceptor sugar is a trisaccharide that is converted to a tetrasaccharide. In other embodiments, the acceptor sugar is a tetrasaccharide that is converted to a pentasaccharide. In some embodiments, the acceptor sugar is a pentasaccharide that is converted to a hexasaccharide. The acceptor sugar may be internalized by the microbial cell by utilizing specific transporters, preferably present in the cytoplasmic membrane of the microbial cell. Additionally and / or alternatively, the acceptor sugar may be synthesized intracellularly by the microbial cell.

[0042] In some embodiments, the acceptor sugar is synthesized intracellularly by the microbial cell from a monosaccharide that is internalized by the microbial cell and extended by a monosaccharide moiety to obtain a disaccharide, which consists of 3, 4, 5, 6 or more monosaccharide moieties and can be further extended by the addition of additional monosaccharide moieties such that an oligosaccharide is synthesized intracellularly that constitutes an acceptor sugar for the biosynthesis of an oligosaccharide of interest. For example, the microbial cell internalizes glucose, which is converted to lactose by the addition of a galactose moiety. Lactose can then be converted to an oligosaccharide, such as, for example, 2'-FL, 3-FL, LNT-II, LNT, LNnT, 3'-SL, 6'-SL, etc. Each of the oligosaccharides may be an oligosaccharide of interest or may constitute an acceptor sugar.

[0043] In some embodiments, the microbial cells are engineered to have an enzyme that catalyzes the conversion of an acceptor sugar, i.e., a disaccharide or oligosaccharide, to an oligosaccharide of interest by transferring a monosaccharide moiety from a donor substrate to the acceptor sugar. Thus, the microbial cells can contain and express at least one recombinant gene that encodes an enzyme that catalyzes the conversion of an acceptor sugar to an oligosaccharide of interest.

[0044] A gene or equivalent functional nucleotide sequence that encodes an enzymatic ability in a microbial cell to add a monosaccharide moiety to an acceptor sugar from a donor substrate can be a homologous nucleotide sequence or a heterologous nucleotide sequence. The term "homologous" as used herein with respect to a nucleotide sequence refers to a nucleotide sequence that is native to the species to which the microbial cell for producing the oligosaccharide of interest belongs. The term "heterologous" as used herein with respect to a nucleotide sequence refers to a nucleotide sequence that is artificially produced or that originates from a species different from the microbial cell for producing the oligosaccharide of interest. A heterologous nucleotide sequence that originates from a species different from the microbial cell for producing the oligosaccharide of interest can originate from a plant, an animal, including a human, a bacterium, an archaea, a fungus or a virus.

[0045] The enzyme that catalyzes the transfer of a monosaccharide moiety to an acceptor molecule is a glycosyltransferase or a transglycosidase. In some embodiments, the glycosyltransferase catalyzes the transfer of a monosaccharide moiety from a nucleotide-activated sugar as a donor substrate to an acceptor sugar. The glycosyltransferase may be selected from the group consisting of galactosyltransferase, glucosaminyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, glucuronosyltransferase, mannosyltransferase, xylosyltransferase, and fucosyltransferase. In further and / or alternative embodiments, the glycosyltransferase is selected from the group consisting of α-1,2-mannosyltransferase, β-1,4-xylosyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,6-N-acetylglucosaminyltransferase, β-1,3-N-acetylgalactosaminyltransferase, β-1,4-N-acetylgalactosaminyltransferase, α-1,3-N-acetylgalactosaminyltransferase, The enzyme is selected from the group consisting of α-1,3-galactosyltransferase, β-1,4-galactosyltransferase, β-1,6-galactosyltransferase, α-1,3-glucosyltransferase, α-1,4-glucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, α-2,8-sialyltransferase, α-1,2-fucosyltransferase and α-1,3 / 1,4-fucosyltransferase.

[0046] It is understood that a fucosyltransferase uses GDP-L-fucose as a donor substrate to transfer a fucose moiety to an acceptor sugar, a sialyltransferase uses CMP-N-acetylneuraminic acid as a donor substrate to transfer N-acetylneuraminic acid to an acceptor sugar, and so forth.

[0047] Nucleotide activated sugars can be synthesized by the engineered microbial cells via a de novo pathway. Additionally and / or alternatively, the microbial cells can use a salvage pathway to provide the nucleotide activated sugars.

[0048] In embodiments in which the donor substrate is synthesized by a de novo pathway, the microbial cell has the enzymes required for the de novo biosynthesis of the nucleotide activated sugar. In some embodiments, the microbial cell is genetically engineered to have at least one recombinant gene encoding the enzymes required for the de novo biosynthetic pathway of the nucleotide activated sugar. In the de novo biosynthetic pathway, the nucleotide activated sugar is synthesized from a "simple" carbon source, such as glucose, sucrose, fructose, or glycerol.

[0049] At least one gene encoding an enzyme required for the de novo biosynthetic pathway of a nucleotide-activated sugar may be endogenous to the microbial cell or may be introduced from an exogenous source into the microbial cell and expressed. The expression, i.e., functional transcription and translation, of the protein encoded by the endogenous gene can be modified by genetically engineering the microbial cell. For example, alteration of the expression of an endogenous gene can be achieved by modifying the transcription promoter of the gene, by modifying the ribosome binding site of the gene, and / or by modifying the codon usage of the protein-encoding nucleotide sequence of the gene. Furthermore, the protein-encoding nucleotide sequence of the gene can be modified such that the activity and / or specificity of the enzyme encoded by the gene is favorable for the desired biosynthetic pathway. Those skilled in the art will know which genes need to be expressed in a genetically modified organism for the de novo synthesis of a nucleotide-activated sugar donor molecule.

[0050] In embodiments in which the salvage pathway is used for the biosynthesis of a nucleotide-activated sugar as a donor substrate, the microbial cell comprises an enzyme that catalyzes the coupling of a nucleotide with a monosaccharide. An example of such an enzyme is the bifunctional fucokinase / L-fucose-1-phosphate-guanylyltransferase FKP from Bacteroides fragilis, which catalyzes a kinase reaction and a pyrophosphatase reaction to form GDP-L-fucose. A further example of an enzyme that catalyzes the coupling of a nucleotide with a monosaccharide is the N-acetylneuraminic acid cytidylyltransferase NeuA from Neisseria meningitidis, which forms CMP-neuraminic acid.

[0051] In some embodiments, the microbial cell having a salvage pathway to provide a nucleotide activated sugar as a donor substrate comprises a recombinant gene encoding an enzyme that catalyzes the coupling of a nucleotide with a monosaccharide for expression of the gene.

[0052] Monosaccharides used as substrates for the formation of nucleotide activated sugars in the salvage pathway can be internalized by microbial cells utilizing monosaccharide transfer proteins. Examples of such monosaccharide transfer proteins are fucose permeases, such as FucP, and sialic acid transporters, such as NanT from E. coli. Monosaccharide transfer proteins can be encoded by and expressed from endogenous genes or encoded by and expressed from recombinant genes.

[0053] In certain embodiments, at least one endogenous gene of the microbial cell that encodes an enzyme or protein that prevents or interferes with the biosynthesis of a nucleotide activated sugar and / or an oligosaccharide of interest is deleted or functionally inactivated.

[0054] In some embodiments, the engineered microbial cell has a sugar transporter in its cell membrane that translocates an oligosaccharide of interest from the cytoplasm of the microbial cell across the cell membrane into the culture medium or, in the case of Gram-negative bacteria, into the periplasmic space.

[0055] Sugar transporters that mediate the translocation of carbohydrates, such as oligosaccharides of interest, across cell membranes may consist of a single polypeptide or may consist of multiple polypeptides (each considered to be a subunit) that form homomeric or heteromeric complexes that translocate the carbohydrate across the cell membrane.

[0056] In some embodiments, the sugar transporter is a member selected from the group consisting of the major facilitator superfamily (MFS), the sugar:cation symporter family, the nucleoside-specific transporter family, the ATP-binding cassette superfamily, and the phosphotransferase system family.

[0057] In certain embodiments, the sugar transporter is encoded by and expressed from a functional recombinant gene. Thus, in such embodiments, the microbial cell comprises a functional gene or operon encoding at least one subunit of a sugar transporter or sugar transporter complex, which facilitates the translocation of intracellularly produced oligosaccharides of interest across the inner membrane of the bacterial cell to its periplasm.

[0058] As used herein, the term "operon" refers to a nucleotide sequence that contains two or more protein coding sequences transcribed from the same regulatory elements to mediate and / or control expression in a microbial cell.

[0059] The term "functional gene" as used herein refers to a nucleic acid molecule that includes a nucleotide sequence that encodes a protein or polypeptide, and also includes a regulatory sequence operably linked to the protein-coding nucleotide sequence (open reading frame) such that the nucleotide sequence encoding the protein or polypeptide can be expressed in / by a cell that harbors the functional gene. Thus, when cultured under conditions that permit expression of the functional gene, the gene is expressed, and cells that express the gene typically contain the protein or polypeptide encoded by the protein coding region of the functional gene. As used herein, the terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and include known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, unless otherwise limited.

[0060] The term "deregulated" as used herein with respect to an endogenous gene refers to altered expression of the protein coding region of the endogenous gene compared to the expression of the gene in the natural precursor of the genetically engineered microbial cell. Expression of a gene can be deregulated by different means known to those skilled in the art. Examples of deregulated expression of a gene include alteration of the natural nucleotide sequence of the promoter of the gene, alteration of the natural nucleotide sequence of the ribosome binding site of the gene. The term "deregulated" includes increased expression of a gene as well as decreased or reduced expression of a gene.

[0061] The term "operably linked" as used herein shall mean a functional linkage between an expression-controlling nucleotide sequence (such as a promoter, a signal sequence, or an array of transcription factor binding sites) and a second nucleotide sequence, where the expression-controlling nucleotide sequence affects the transcription and / or translation of the second nucleotide sequence. Thus, the term "promoter" refers to a nucleotide sequence that usually "precedes" a protein-coding nucleotide sequence in a DNA polynucleotide and provides a site for the initiation of transcription into mRNA. "Regulatory" DNA sequences are also protein-coding nucleotide sequences that usually "upstream" (i.e., precede) a gene in a given DNA polymer and bind to proteins that determine the frequency (or rate) of transcription initiation. Collectively referred to as "promoter / regulatory" or "control" DNA sequences, these sequences preceding a selected gene (or set of genes) in a functional DNA polymer cooperate to determine whether transcription (and ultimately expression) of the gene occurs. Nucleotide sequences that "follow" a protein-coding nucleotide sequence in a DNA polymer and provide a signal for the termination of transcription into mRNA are referred to as transcription "termination" sequences.

[0062] The term "recombinant" as used herein indicates that a polynucleotide, such as a gene or operon, has been produced by genetic engineering. Thus, a recombinant gene, operon or polynucleotide does not naturally occur in a cell of the same species as the Gram-negative bacterial cell for producing the desired oligosaccharide. A recombinant polynucleotide may contain a heterologous nucleotide sequence (i.e., a nucleotide sequence that is foreign to the microbial cell) or express a peptide or protein encoded by a heterologous nucleotide sequence. A recombinant microbial cell may contain a gene not found in its native (non-recombinant) predecessor. A recombinant cell may also contain a variant of a gene found in the recombinant cell's natural predecessor, where the gene has been modified and reintroduced into the cell by technical means. The term "recombinant" also encompasses bacterial cells, which include nucleotide sequences that are endogenous to the bacterial cell and have been modified without removing the nucleic acid molecule having the nucleotide sequence from the bacterial cell. Such modifications include those obtained by gene replacement, site-directed mutagenesis and related techniques. Thus, a "recombinant polypeptide" is one produced by a recombinant cell.

[0063] As used herein, a "heterologous nucleotide sequence" or "heterologous nucleic acid" is one that originates from a source foreign to a particular host cell (e.g., originates from a different species) or, if originating from the same source, has been modified from its original form. Thus, a heterologous nucleotide sequence may be a nucleotide sequence in which a nucleotide sequence encoding a heterologous protein is operably linked to a promoter, where the nucleotide sequence encoding the protein and the nucleotide sequence of the promoter are obtained from different source organisms, or, if from the same source organism, the nucleotide sequence encoding the protein or the promoter has been modified from its original form. A heterologous nucleotide sequence may be stably introduced into the genome of a bacterial cell, for example, by transposition, transfection, transformation, conjugation or transduction. The techniques that may be applied depend on the specificity of the bacterial cell and the nucleic acid molecule to be introduced into the bacterial cell. 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 Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Thus, a "genetically engineered bacterial cell" or a "genetically engineered microbial cell" is understood as a bacterial cell that has been transformed or transfected or is capable of being transformed or transfected by an exogenous polynucleotide sequence. Thus, the nucleotide sequence used in the present invention may be, for example, contained in a vector that is stably transformed / transfected or otherwise introduced into the host microbial cell. A wide variety of expression systems can be used to produce the polypeptide. Such vectors include, inter alia, chromosomal, episomal and viral derived vectors, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viral derived vectors, and combinations thereof, such as vectors derived from plasmids and bacteriophage genetic elements, such as cosmids and phagemids.The expression system construct may include control regions that regulate developmental as well as developmental expression. In this regard, generally, any system or vector suitable for maintaining, propagating or expressing polynucleotides and synthesizing polypeptides in bacterial host cells may be used for expression. The appropriate nucleotide sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those described in Sambrook et al., supra. Preferably, the polynucleotide comprising the recombinant nucleotide sequence is stably introduced into the genome of the microbial cell. Genomic integration may be achieved by recombination or transposition.

[0064] The functional gene or genes encoding a transporter or transporter complex that facilitates transport of the oligosaccharide of interest across the inner membrane can be either endogenous or exogenous to the engineered microbial cell.

[0065] In some embodiments, the sugar transporter gene is endogenous to the genetically engineered bacterial cell. The functional elements that direct the synthesis of the protein encoded by the open reading frame of the sugar transporter gene may be modified such that the level of transcription of the open reading frame or translation of the mRNA that leads to the synthesis of the transporter protein is different from that naturally found in the preceding cell. The modification of the functional element can increase the transcription and / or translation level or decrease the transcription and / or translation level compared to the unmodified state. The modification can be the modification of a single nucleotide or the replacement of the complete functional element. In either case, the non-native level of transcription and / or translation is optimized for the transport of the intracellular oligosaccharide of interest across the inner membrane.

[0066] In another embodiment, the gene or genes encoding the sugar transporter or transporter complex are exogenous to the engineered microbial cell. In such an embodiment, the protein coding region is operably linked to naturally occurring or artificial functional elements, such as promoters or ribosome binding sites, such that the transcription and translation levels of the transporter are optimized for transport of the oligosaccharide of interest across the inner membrane.

[0067] Transcription of the gene encoding the sugar transporter or a subunit of the sugar transporter can be constitutive or regulated. The skilled artisan knows how to select and use a promoter and / or a promoter in combination with further transcriptional regulators to achieve optimal expression of the gene encoding the sugar transporter.

[0068] In embodiments where the gene encoding the sugar transporter is heterologous to the microbial cell for production of an oligosaccharide of interest, the nucleotide sequence of the gene may be modified compared to the naturally occurring nucleotide sequence, but may encode a polypeptide having an unmodified amino acid sequence.

[0069] A sugar transporter for translocating an oligosaccharide of interest across a cell membrane can have its native amino acid sequence, i.e., the amino acid sequence found in nature. Alternatively, the amino acid sequence of the sugar transporter can be modified compared to its native amino acid sequence such that the resulting variant has enhanced activity with respect to translocating the oligosaccharide of interest and / or other beneficial characteristics, such as enhanced protein stability or more stable integration into the inner membrane.

[0070] In certain embodiments, the nucleotide sequence encoding the sugar transport protein is artificial, and therefore the amino acid sequence of the resulting protein that acts as a transporter to transport an oligosaccharide of interest from the cytoplasm to the periplasm is not found in nature.

[0071] The term "variant" as used herein refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential (enzymatic) properties of the reference polynucleotide or polypeptide. A typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide. A change in the nucleotide sequence of a variant may or may not change the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in a polypeptide encoded by a reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring variant, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques, direct synthesis, and other recombinant methods known to those of skill in the art.

[0072] Within the scope of the present invention, nucleic acid / polynucleotide and polypeptide polymorphic variants, alleles, mutants and interspecies homologs are also constituted by those terms having an amino acid sequence having greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more amino acid sequence identity to a polypeptide encoded by a wild-type protein, preferably identity over a region of at least about 25, 50, 100, 200, 500, 1000 or more amino acids.

[0073] A "variant" of any gene / protein is meant to refer to a sequence variant of the gene / protein that retains the same, lower or higher activity of the gene, protein and / or protein encoded by the gene, or a different activity with respect to the substrate specificity of the protein, or a different activity with respect to the reaction specificity of the protein from which the variant is derived.

[0074] With respect to enzymes directly involved in the pantothenic acid biosynthetic pathway, "functional variant" is meant to denote a sequence variant of the enzyme and / or the gene encoding the enzyme that is identical to the respective gene / enzyme from which the variant is derived, but retains reduced activity.

[0075] The microbial cells genetically engineered to produce the desired oligosaccharides either lack pantothenic acid intracellular biosynthesis or have low levels of intracellular pantothenic acid biosynthesis.

[0076] In some embodiments, the engineered microbial cells for producing the oligosaccharide of interest are unable to synthesize pantothenic acid endogenously. In additional and / or alternative embodiments, the engineered microbial cells are engineered such that they are unable to synthesize pantothenic acid endogenously, i.e., the pantothenic acid biosynthetic pathway within the microbial cell is disrupted.

[0077] Disruption of the pantothenic acid biosynthetic pathway can be achieved by deleting or functionally inactivating at least one of the genes of the microbial cell that encodes an enzyme directly involved in the pantothenic acid biosynthetic pathway, i.e., in one of the subsequent enzymatic reactions that lead to the intracellular formation of pantothenic acid from any given educt. Preferably, the enzyme catalyzes only reactions exclusive to the pantothenic acid biosynthetic pathway.

[0078] The at least one gene encoding an enzyme directly involved in the pantothenic acid biosynthetic pathway may be selected from the group of genes encoding a ketopantoate hydroxymethyltransferase, e.g., panB, a pantothenate synthetase, e.g., panC, a ketopantoate reductase, e.g., panE, and an acetohydroxyacid isomeroreductase, e.g., ivfC.

[0079] At least one gene encoding an enzyme directly involved in the pathway of pantothenic acid biosynthesis may be deleted in its entirety or a substantial part of this gene may be deleted. The term "substantial" with respect to a part of at least one gene refers to the nucleotide sequence of the gene essential for the expression and / or activity of the enzyme encoded by this gene. A "substantial part" of a gene in the sense of the present disclosure is understood to include expression control sequences, such as promoters, and / or the protein coding region of the gene, which encodes an amino acid sequence whose presence in the enzyme is essential for the enzymatic activity of the enzyme. Thus, the expression of the gene and / or the enzymatic activity of the polypeptide encoded by the gene is lost.

[0080] The functional inactivation of at least one gene encoding an enzyme directly involved in the pantothenic acid biosynthetic pathway can be achieved by replacing at least one codon of the gene with a codon encoding an amino acid different from the replaced codon, such that the polypeptide encoded by the gene thus modified does not have the enzymatic activity of the pantothenic acid biosynthetic pathway. In another embodiment, the codon of the gene encoding an enzyme directly involved in the pantothenic acid biosynthetic pathway is replaced with a stop codon, such that the gene thus modified encodes a fragment of a native enzyme that does not have the enzymatic activity of the pantothenic acid biosynthetic pathway.

[0081] In yet other embodiments, at least one gene encoding an enzyme directly involved in the pantothenic acid biosynthetic pathway is functionally inactivated in that a frameshift mutation is inserted into the protein coding region of the gene such that the polypeptide encoded by the thus modified gene does not exhibit enzymatic activity of the pantothenic acid biosynthetic pathway.

[0082] Pantothenic acid is a precursor of coenzyme A. Coenzyme A is an essential compound for the metabolism of cells. In embodiments where the expression or activity of at least one of the enzymes involved in the direct pantothenic acid biosynthesis pathway is lost, the microbial cells are unable to synthesize pantothenic acid. Such microbial cells require the internalization of exogenous pantothenic acid. Thus, microbial cells in which the expression or activity of at least one of the enzymes directly involved in the pantothenic acid biosynthesis pathway is lost have a pantothenic acid permease for the internalization of exogenous pantothenic acid. Having a pantothenic acid permease allows the microbial cells to internalize exogenous pantothenic acid, grow, and produce the oligosaccharides of interest, despite the inability to synthesize pantothenic acid.

[0083] In some embodiments, microbial cells for producing a target oligosaccharide that are unable to synthesize pantothenic acid are genetically engineered to harbor and express a gene encoding a pantothenate permease.

[0084] A suitable pantothenate permease is E. coli PanF. Thus, in some embodiments, the microbial cell for producing the oligosaccharide of interest contains and expresses a gene encoding E. coli PanF or a functional variant thereof, preferably an E. coli panF gene or a functional variant thereof.

[0085] Cultivating microbial cells that cannot synthesize pantothenic acid but can internalize exogenous pantothenic acid in the presence of exogenous pantothenic acid is advantageous because the amount of pantothenic acid in the culture medium can be adjusted so that the microbial cells can grow and synthesize the desired oligosaccharide in versatile amounts, but can avoid the presence of excess pantothenic acid in the culture medium at the end of the fermentative production process of the desired oligosaccharide.

[0086] In some embodiments, the microbial cells engineered to produce an oligosaccharide of interest have low levels of intracellular pantothenic acid biosynthesis, hi some embodiments, the microbial cells are engineered to have impaired expression and / or activity of at least one enzyme directly involved in the pantothenic acid biosynthetic pathway.

[0087] The term "impaired" as used herein means that the expression and / or activity of at least one enzyme directly involved in pantothenic acid biosynthesis is reduced compared to the expression and / or activity of the enzyme in a microbial cell of a precursor cell line that has not been genetically engineered to have a reduced level of pantothenic acid biosynthesis and thus a low level of pantothenic acid biosynthesis.

[0088] The expression "reduced" with respect to the level of intracellular pantothenic acid biosynthesis is understood as the level of intracellular pantothenic acid biosynthesis compared to wild-type microbial cells, i.e. the type of microbial cell of the same genus that occurs most commonly in nature, in particular when the microbial cells are cells that have not been obtained by genetic engineering to reduce intracellular pantothenic acid biosynthesis, but have been obtained, for example, by selection with or without prior random mutagenesis, or, when the microbial cells have been genetically engineered to impair the level of pantothenic acid biosynthesis, compared to the cells of a precursor cell line that have not been genetically engineered to have a reduced intracellular level of pantothenic acid biosynthesis. When comparing the levels of intracellular pantothenic acid biosynthesis, the genetically engineered microbial cells are substantially identical to the cells of the precursor cell line in terms of genotype, apart from the genetic information being engineered to reduce the level of intracellular pantothenic acid biosynthesis, nevertheless, the microbial cells as well as the cells of the precursor cell line may be genetically engineered microbial cells that have been genetically engineered for purposes other than impairing intracellular pantothenic acid biosynthesis, for example engineered to be able to synthesize an oligosaccharide of interest.

[0089] The level of pantothenic acid biosynthesis in the genetically engineered microbial cells with impaired or reduced pantothenic acid biosynthesis is reduced such that the amount of pantothenic acid in the culture medium is reduced by at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500-fold. It is understood that the reduction in pantothenic acid in the culture medium is evaluated by inoculating and incubating the culture of the microbial cells with reduced levels of pantothenic acid biosynthesis and the culture of the comparative microbial cells under substantially identical conditions, e.g., with respect to the growth stage and cell density in the preculture, the volume of medium inoculated, the medium composition, the amount of preculture / number of cells used to inoculate the main culture, the culture conditions, e.g., temperature, shaking speed, time / cell density at which samples are taken to measure the amount of pantothenic acid in the culture medium, etc. Preferably, the reduced level of pantothenic acid in the culture medium is present at the end of the fermentative production of the oligosaccharide of interest.

[0090] In some embodiments, the level of pantothenic acid is reduced in that the expression of at least one gene encoding an enzyme directly involved in the biosynthetic pathway of pantothenic acid is reduced. The expression of a gene can be reduced in that the nucleotide sequence of at least one expression control sequence of the gene is altered such that the expression of the protein coding region of the enzyme is reduced. An example of changing the nucleotide sequence of an expression control sequence to reduce the expression of a gene encoding an enzyme directly involved in the biosynthetic pathway of pantothenic acid includes the deletion of one or more nucleotides, the insertion or exchange of one or more nucleotides in the expression control sequence of the native gene. Another example of changing the nucleotide sequence of an expression control sequence to reduce the expression of a gene encoding an enzyme directly involved in the biosynthetic pathway of pantothenic acid includes replacing a given promoter with another promoter, the other promoter being a weaker promoter than the preceding promoter. Considering that the strength of the promoter is known or can be determined by assays known in the art, it is possible to replace the native promoter of the gene with a heterologous promoter, which accounts for the weaker expression of the protein coding region of the resulting recombinant gene.

[0091] In some embodiments, the level of translation, i.e., the level of translation of mRNA into an amino acid sequence, is compared to the level of translation found in a wild-type cell or a progenitor cell. The level of translation can be altered by changing one or more nucleotides within the ribosome binding site and / or within the nucleotide sequence upstream of the ribosome binding site, thereby affecting and decreasing translation compared to the level found in the unaltered state.

[0092] In further and / or alternative embodiments, the microbial cell comprises a gene encoding a variant of an enzyme directly involved in the pantothenic acid biosynthetic pathway, the variant exhibiting reduced enzymatic activity. In some embodiments, the protein coding region of the gene has been modified to encode the variant. In some embodiments, the microbial cell comprises a heterologous gene encoding a variant of an enzyme directly involved in the pantothenic acid biosynthetic pathway, the endogenous gene being deleted or functionally inactivated.

[0093] In further and / or alternative embodiments, the microbial cell has a higher K for its substrate compared to the native enzyme. m The mutants of enzymes directly involved in the pantothenic acid biosynthesis pathway have a lower Michaelis constant (i.e., lower activity). m The value is the K m Preferably, the increase is at least 100-fold, more preferably at least 200-fold, and most preferably at least 500-fold compared to the value.

[0094] K of enzymes directly involved in the pantothenic acid biosynthesis pathway m The value can be increased in that at least one amino acid of the enzyme involved in the catalytic activity / substrate binding of the enzyme is replaced with a different amino acid. The amino acid sequence of the enzyme can be modified such that one or more codons in the protein coding region of the gene encoding the enzyme directly involved in the pantothenic acid biosynthetic pathway are replaced with codons encoding a different amino acid.

[0095] Instead, mutants of enzymes directly involved in pantothenic acid biosynthesis have at least one amino acid in the amino acid sequence involved in catalytic activity, which is expressed as a k cat The enzyme can be modified relative to the wild type enzyme by exchanging it to have a lower k cat Preferably the value is 10-fold, 100-fold or 500-fold lower compared to the wild-type enzyme.

[0096] In some embodiments, endogenous genes encoding enzymes directly involved in the pantothenic acid biosynthetic pathway are altered, in alternative embodiments, the endogenous genes are deleted or functionally inactivated and mutant enzymes are expressed from recombinant genes, e.g., as described herein above.

[0097] In certain embodiments, the microbial cell lacks its endogenous ketopantoate reductase and is capable of converting the ketopantoate K of E. coli ketopantoate reductase PanE having the amino acid sequence set forth in SEQ ID NO:1. m The K of ketopantoate is at least 100 times, preferably at least 200 times, and more preferably at least 500 times higher than the K m The Michaelis-Menten constant (K m ) is defined as the substrate concentration at which the reaction rate is half its maximum (in other words, it defines the substrate concentration at which half of the active sites are occupied).

[0098] In some embodiments, the K for ketopantoate is higher than that of E. coli ketopantothenate reductase having the amino acid sequence set forth in SEQ ID NO:1. m Ketopantoate reductase with increased K m It is a mutant of E. coli PanE with a K value. m Preferably, the value is increased by at least 100-fold, more preferably by at least 200-fold, and most preferably by at least 500-fold.

[0099] In further and / or alternative embodiments, increased K for ketopantoate mThe ketopantoate reductase having the value is a variant of the E. coli K-12 PanE enzyme having an amino acid containing a pure carbohydrate side chain at positions 98, 176, 244 and / or 256 with respect to the amino acid sequence set forth in SEQ ID NO: 1. The amino acid containing a pure carbohydrate side chain is an amino acid selected from the group consisting of glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, F) and proline (Pro, P).

[0100] In certain embodiments, the increased K for ketopantoate m The mutants of ketopantoate reductase having a K value are selected from the group of mutants consisting of E. coli K-12 PanE(N98A), E. coli K-12 PanE(K176A), E. coli K-12 PanE(S244A), E. coli K-12 PanE(E256A), and combinations of these variations with the provision of mutants E. coli K-12(K176A, E256A) and other mutants lacking ketopantoate reductase activity. The K values ​​of ketopantoate caused by different variations are shown in Table 1. m The increase in values ​​is shown in Table 1.

[0101] [Table 1]

[0102] Microbial cells with a low level of intracellular pantothenic acid biosynthesis are advantageous for use in the production of oligosaccharides of interest because they can synthesize pantothenic acid intracellularly and therefore do not require exogenous pantothenic acid supply during cultivation, but the amount of pantothenic acid in the medium at the end of the fermentation process to produce the oligosaccharides of interest is low. The low level of pantothenic acid in the culture medium allows for enhanced recovery of the oligosaccharides of interest.

[0103] According to a second aspect, there is provided a use of a genetically engineered microbial cell having low levels or no pantothenic acid biosynthesis as disclosed herein to produce an oligosaccharide of interest, which synthesizes the oligosaccharide of interest when cultured in a medium under conditions that allow the microbial cell to synthesize the oligosaccharide of interest.

[0104] Utilizing these genetically engineered microbial cells to produce oligosaccharides of interest is advantageous over the use of microbial cells that do not have low levels of pantothenic acid biosynthesis, because the amount of pantothenic acid in the fermentation broth at the end of the culture step is much lower. Preferably, the amount of pantothenic acid in the fermentation broth at the end of the fermentation step is so low that efforts to subsequently purify the oligosaccharides of interest from the fermentation broth do not require one or more process steps tailored to remove pantothenic acid from the fermentation broth or from a process stream derived from the fermentation broth.

[0105] Surprisingly, it has been found that engineered microbial cells with low levels of pantothenic acid biosynthesis exhibit higher production of oligosaccharides of interest compared to cells of their progenitor cell line that have also been engineered to have low levels of pantothenic acid biosynthesis.

[0106] In some embodiments, production of the oligosaccharide of interest is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to production of the oligosaccharide of interest by cells of those progenitor cell lines that have not been genetically engineered to have low levels of pantothenic acid biosynthesis.

[0107] According to a third aspect, there is provided a method for producing an oligosaccharide of interest in a genetically engineered microbial cell, the method comprising: - Providing a genetically engineered microbial cell with a metabolic pathway for the intracellular synthesis of an oligosaccharide of interest, with or without low levels of pantothenic acid biosynthesis, - culturing the genetically engineered microbial cells in a culture medium under conditions that allow the microbial cells to synthesize the oligosaccharide of interest intracellularly; and - Optionally recover the desired oligosaccharides. Includes.

[0108] The genetically engineered microbial cell having a metabolic pathway for the intracellular synthesis of an oligosaccharide of interest, with or without low levels of pantothenic acid biosynthesis, is preferably a microbial cell as described herein above.

[0109] The oligosaccharides of interest are those oligosaccharides previously described herein, preferably HMOs.

[0110] In some embodiments, preferably in embodiments where the genetically engineered microbial cells do not have pantothenic acid biosynthesis, the genetically engineered microbial cells are cultured in the presence of exogenous pantothenic acid. It is understood, however, that pantothenic acid is added to the culture medium before and / or during the culture of the genetically engineered microbial cells. The supply of exogenous pantothenic acid to the culture medium can be adjusted according to predetermined process parameters and / or given requirements. Before the end of the fermentation production process for producing the oligosaccharide of interest, the supply of exogenous pantothenic acid to the culture medium is stopped so that at the end of the fermentation process for synthesizing the oligosaccharide of interest, the culture medium contains less than 500 μg / ml, 400 μg / ml, 300 μg / ml, 200 μg / ml, 100 μg / ml, 80 μg / ml, 60 μg / ml or even less than 40 μg / ml.

[0111] According to a fourth aspect, there is provided a preparation of an oligosaccharide of interest, preferably a HMO as described hereinbefore, which preparation is obtainable by using a microbial cell or by using a method for producing an oligosaccharide of interest as described hereinbefore.

[0112] The preparation of the oligosaccharide of interest may be used to manufacture a nutritional composition. The nutritional composition may be a dietary supplement, a medicinal food, an infant formula, or any other food product that is restricted with respect to the maximum amount of pantothenic acid contained therein. The nutritional composition preferably contains an amount of the oligosaccharide of interest substantially similar to the amount of the oligosaccharide of interest naturally occurring in human milk. The use of the preparation of the oligosaccharide of interest for the manufacture of a nutritional composition, particularly an infant formula, is advantageous because it reduces the risk of exceeding the upper limit of pantothenic acid formulated in such a nutritional composition and does not require costly processing steps to remove pantothenic acid during the production of the oligosaccharide of interest, nor adjustments to the existing manufacturing process of the nutritional composition.

[0113] The present invention also extends to nutritional compositions containing one or more oligosaccharides of interest produced by using genetically engineered microbial cells and / or methods of producing the oligosaccharides of interest as described herein above.

[0114] The present invention will be described with reference to certain embodiments and with reference to the drawings, but the present invention is not limited thereto, but only by the claims. Moreover, terms such as first, second, etc. in this specification and claims are used to distinguish between similar elements and are not necessarily intended to describe an order in time, space, ranking, or otherwise. It will be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein can operate in an order other than that described or illustrated herein.

[0115] It should be noted that the term "comprising" used in the claims should not be interpreted as being limited to the means listed thereafter, nor should it exclude other elements or steps. It should therefore be interpreted as specifying the presence of the mentioned and described features, integers, steps or components, but without excluding the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting only of components A and B. This means that in the context of the present invention, the only relevant components of the device are A and B.

[0116] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0117] Similarly, in describing exemplary embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and facilitating understanding of one or more of the various aspects of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects include less than all features of a single foregoing disclosed embodiment. Thus, the claims following the Detailed Description are expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment of the invention.

[0118] Furthermore, some embodiments described herein include some features that are included in other embodiments but do not include other features, meaning that combinations of features of different embodiments form different embodiments within the scope of the present invention and as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0119] Additionally, some of the embodiments are described herein as methods or combinations of elements of methods that may be implemented by a processor of a computer system or by other means for carrying out the functions. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Additionally, the elements described herein of the apparatus embodiments are examples of means for carrying out the functions performed by the elements for carrying out the invention.

[0120] In the description and drawings provided herein, numerous specific details are set forth. However, it is understood that embodiments of the present invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0121] The present invention will now be described by detailed descriptions of some embodiments of the present invention. Other embodiments of the present invention can be constructed according to the knowledge of those skilled in the art without departing from the true spirit or technical teaching of the present invention, and it is apparent that the present invention is limited only by the terms of the appended claims. EXAMPLES

[0122] LC / MS analysis The identity of the products was determined by multiple reaction monitoring (MRM) using an LC triple quadrupole MS detection system (Shimadzu LCMS-8050). Precursor ions were selected and analyzed in quadrupole 1, followed by collision-induced fragmentation (CID) with argon and selection of fragment ions in quadrupole 3. Selected transitions and collision energies of intermediate and end-product metabolites are listed in Table 2. LNT, 3'-SL and pantothenic acid (vitamin B5) in particle-free culture supernatants or cytoplasmic fractions were separated by high-performance liquid chromatography (HPLC) using a Waters XBridge BEH Amide HPLC column (3.5 μm, 2.1 × 50 mm) protected with an XBridge BEH Amide VanGuard cartridge (3.9 × 5 mm) for both neutral or acidic sugars. Neutral sugars were eluted with an isocratic flow of HO containing 0.1% (v / v) ammonium hydroxide. Prior to LC / MS analysis, samples of neutral oligosaccharides were prepared by filtration (0.22 μm pore size) and clarified by solid-phase extraction on Strata ABW ion exchange matrix (Phenomenex). Samples containing 3'-sialyllactose and pantothenic acid were filtered through a 0.22 μm filter, heated at 95°C for 5 min, and centrifuged at 13,000 x g for 5 min. HPLC separation of acidic sugars was performed using gradient elution with buffer A consisting of 50% acetonitrile and buffer B consisting of 80% acetonitrile, both containing 10 mM ammonium acetate at pH 4.2. The HPLC system was a Shimadzu Nexera X2 SIL-30AC operated at 8°C for elution of neutral sugars. MPThe instrument contained an autosampler, a Shimadzu LC-20AD pump, and a Shimadzu CTO-20AC column oven operated at 35 °C and adjusted to 50 °C for the elution of acidic sugars. 1 μl was injected into the instrument for LNT analysis and 0.5 μL for 3'-SL and pantothenic acid measurements. The flow rates were set at 0,3 mL / min (neutral), 0,4 mL / min (acidic), corresponding to a run time of 5 min. Meanwhile, LNT was analyzed in negative ion mode, while 3'-SL and pantothenic acid were examined in positive ion mode. The mass spectrometer was operated at unit resolution. Collision energy, Q1 and Q3 pre-bias were optimized for each analyte. Quantitation methods were established using commercially available standards (Carbosynth, Elicityl, Sigma Aldrich).

[0123] [Table 2]

[0124] Culture medium The culture medium used to grow the cells to produce the desired oligosaccharides contained 3 g / L KH2PO4, 12 g / L K2HPO4, 5 g / L (NH4)SO4, 0.3 g / L citric acid, 0.1 g / L NaCl, 2 g / L MgSO4 x 7 H2O, and 0.015 g / L CaCl2 x 6 H2O, supplemented with 1 ml / L trace element solution (54.4 g / L ferric ammonium citrate, 9.8 g / L MnCl2 x 4 H2O, 1.6 g / L CoCl2 x 6 H2O, 1 g / L CuCl2 x 2 H2O, 1.9 g / L MnCl2 x 4 H2O, 1.1 g / L Na2MoO4 x 2 H2O, 1.5 g / L Na2SeO3, 1.5 g / L NiSO4 x 6 H2O). The pH of the medium was adjusted to 7.0. For the synthesis of the desired oligosaccharides, lactose was added to the medium.

[0125] [Example 1] Metabolic engineering of E. coli to reduce pantothenic acid biosynthesis. To introduce a mutation in the panE gene resulting in the amino acid exchange S244A in E. coli PanE, an antibiotic resistance cassette was inserted between the genes thiI and yjaL by homologous recombination. The antibiotic resistance cassette was flanked by 640 nucleotides representing the 3' end of thiI and 777 nucleotides including the 3' end of yjaL and panE. The flanking regions were amplified from the genomic DNA of E. coli BL21(DE3). While amplifying yjaL-'panE, two single point mutations were introduced resulting in an amino acid exchange from serine to alanine at position 244 of the PanE protein. Homologous recombination was performed and promoted by the expression of the lambda Red genes gam, beta and exo from the plasmid pKD46 according to Datsenko and Wanner (Proc. Natl. Acad. Sci. (2000) 97, 6640-66645 (2000)). The mutation was confirmed by sequencing the panE gene in the E. coli genome.

[0126] [Example 2] Preparation of sialyllactose with reduced pantothenic acid content A metabolically engineered derivative of E. coli BL21(DE3), capable of synthesizing 3'-sialyllactose intracellularly, was genetically engineered to reduce pantothenic acid biosynthesis.

[0127] The strain was grown in mineral salts medium using glycerol as the sole carbon source. 200 ml of preculture was inoculated and grown for 24 h at 30° C. under aerobic conditions. This preculture was used to inoculate a fermenter containing 1 L of mineral salts medium and 2% glycerol (OD 600 The medium was inoculated to an optical density of 0.15. The fermentation was carried out at 30°C, pH 7.0, under aerobic conditions. The OD of approximately 5 was reached. 600 The cultures were fed with 100 mM lactose before glycerol was consumed from the culture medium. Once in the fed-batch phase, glycerol was fed continuously and the cells were grown under C-source limitation. The concentrations of 3'-SL and pantothenate in the culture supernatants were measured.

[0128] FIG. 3 shows the growth of bacterial cell cultures (◆) and the amount of pantothenic acid in the culture medium of the bacterial cells (□), with the graph in FIG. 3A showing the growth and pantothenic acid amount of a parent E. coli strain and the graph in FIG. 3B showing the growth and pantothenic acid amount of an E. coli strain genetically engineered to have reduced or low levels of pantothenic acid biosynthesis.

[0129] After 95 hours of fermentation, a 1.15-fold higher 3'-SL titer was achieved using the E. coli strain engineered to have reduced levels of pantothenic acid biosynthesis than the 3'-SL titer of the parent strain grown under the same conditions, and the pantothenic acid level in the culture supernatant was 0.01-fold higher than the pantothenic acid level in the culture supernatant of the parent strain grown under the same conditions.

[0130] [Example 3] Preparation of pantothenic acid-reduced lacto-N-tetraose A genetically engineered derivative of the E. coli strain BL21(DE3), capable of synthesizing lacto-N-tetraose intracellularly, was further engineered to have reduced pantothenic acid biosynthesis. The endogenous panE gene of the derivative was replaced with a mutant encoding PanE S244A.

[0131] The latter LNT-producing E. coli strain was grown in mineral salts medium using glycerol as the sole carbon source. Four precultures of 200 ml were inoculated and grown for 40 h at 30° C. under aerobic conditions. These precultures were used to seed a fermenter containing 180 L of mineral salts medium to reach an OD 600 This well-growing seed culture was used to inoculate a production fermenter containing 2500 L of mineral salts medium supplemented with 60 mM lactose, 5 g / L NH4Cl, and 2% glycerol to an optical density of OD = 0.1. 600The cells were inoculated to an optical density of 0.1. The cells were cultivated at 30°C, pH 6.8, under aerobic conditions. Once the culture entered the fed-batch phase, glycerol was continuously fed and the bacteria were further grown under C-source limitation. Once the dissolved oxygen level was stable, continuous lactose feeding was started to keep the lactose concentration in the medium at a sufficient level for LNT production. The concentrations of LNT and pantothenate in the culture supernatant were measured.

[0132] After 86 h of fermentation, the concentration of pantothenic acid in the culture supernatant was more than 16-fold lower compared to the supernatant of the parental cell culture. Surprisingly, the amount of LNT in the culture supernatant increased more than 1.4-fold in cultures of E. coli strains with reduced pantothenic acid biosynthesis compared to the parental strain.

[0133] FIG. 4 shows the growth of bacterial cell cultures (◆) and the amount of pantothenic acid in the culture medium of the bacterial cells (□), with the graph in FIG. 4A showing the growth and pantothenic acid amount of a parent E. coli strain and the graph in FIG. 4B showing the growth and pantothenic acid amount of an E. coli strain genetically engineered to have reduced or low levels of pantothenic acid biosynthesis.

Claims

1. A genetically engineered microbial cell for producing a desired oligosaccharide, the genetically engineered microbial cell having a metabolic pathway for intracellular biosynthesis of the desired oligosaccharide and having low levels or no intracellular biosynthesis of pantothenic acid.

2. The oligosaccharide of interest is selected from the group of human milk oligosaccharides, preferably 2'-fucosyl-lactose (2'-FL), 3-fucosyl-lactose (3-FL), 2',3-difucosyllactose (DFL), lacto-N-triose II, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucco-pentaose I (LNnFP-I), lacto-N-fucopentaose II (LNFP-II ... to-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucco-pentaose V (LNnFP-V), lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto-N-difucosylhexaose I, Lacto-N-difucosyl-hexaose II, para-lacto-N-fucosyl-hexaose (paraLNH), fucosyl-lacto-N-sialylpentaose a (F-LST-a), fucosyl-lacto-N-sialylpentaose b (F-LST-b), fucosyl-lacto-N-sialylpentaose c (F-LST-c), fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose (3F-3'-SL) 3-Fucosyl-6'-sialyllactose (3F-6'-SL), lacto-N-neodifuco-hexaose I, 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c), disialyl-lacto-N-tetraose (DS-LNT), disialyl-lacto-N-fuco-pentaose (DS-LNFP V), lacto-N-neodifucohexaose I (LNnDFH I), 3'-galactosyllactose (3'-GL), 6'-galactosyllactose (6'-GL).

3. 3. The genetically engineered microbial cell of claim 1 or 2, wherein endogenous genes encoding enzymes directly involved in the pantothenic acid biosynthetic pathway have been deleted or functionally inactivated.

4. 3. The genetically engineered microbial cell of claim 1 or 2, wherein the low level of pantothenic acid biosynthesis results in at least a 10-fold reduction in the amount of pantothenic acid in the culture medium.

5. 5. The genetically engineered microbial cell according to any one of claims 1, 2 and 4, wherein the expression and / or activity of at least one enzyme directly involved in the intracellular pantothenic acid biosynthetic pathway is impaired.

6. 6. The genetically engineered microbial cell of any one of claims 1, 2, 4 and 5, wherein the at least one enzyme is selected from the group consisting of ketopantoate hydroxymethyltransferase, ketopantoate reductase, acetohydroxyacid isomeroreductase, aspartate 1-decarboxylase and pantothenate synthetase.

7. The ketopantoate reductase has a K of ketopantoate that is preferably increased by at least 100-fold, more preferably at least 200-fold, and most preferably at least 500-fold, compared to E. coli K-12 ketopantoate reductase. ｍ 7. The genetically engineered microbial cell of claim 6, having a CpG2 value.

8. 8. The genetically engineered microbial cell of claim 6 or 7, wherein the ketopantoate reductase is a mutant of E. coli K-12 ketopantoate reductase, said mutant being selected from the group consisting of E. coli K-12 PanE(N98A), E. coli K-12 PanE(K176A), E. coli K-12 PanE(S244A), E. coli K-12 PanE(E256A) and combinations of said mutations.

9. Use of a genetically engineered microbial cell according to any one of claims 1 to 9 for the production of an oligosaccharide of interest.

10. 1. A method for producing an oligosaccharide of interest, said method comprising: - providing a genetically engineered microbial cell that has a metabolic pathway for the intracellular biosynthesis of an oligosaccharide of interest and has low levels or no intracellular biosynthesis of pantothenic acid; - culturing said genetically engineered microbial cells in a medium under conditions that allow the intracellular biosynthesis of said oligosaccharide of interest; and - optionally recovering said oligosaccharides of interest. A method comprising:

11. 11. The method of claim 10, wherein the genetically engineered microbial cells are cultured in the presence of exogenous pantothenic acid.

12. Use of an oligosaccharide preparation obtained by the use of a genetically engineered microbial cell according to any one of claims 1 to 8 or by the method according to any one of claims 10 and 11 for the manufacture of a nutritional composition, preferably for the manufacture of an infant formula.

Images