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CN-122029274-A - Cell production of oligosaccharides comprising lacto-N-trisaccharide (LN 3)

CN122029274ACN 122029274 ACN122029274 ACN 122029274ACN-122029274-A

Abstract

The invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the invention is in the technical field of culturing genetically engineered cells. The invention describes (i) a method for producing oligosaccharides comprising milk-N-trisaccharide (LN 3) or oligosaccharide mixtures comprising oligosaccharides comprising LN3 by culturing genetically modified cells comprising a transporter, and (ii) genetically modified cells for use in said method.

Inventors

  • S. Esalt
  • J. Boplez
  • A. Wacotland

Assignees

  • 因比奥斯公司

Dates

Publication Date
20260512
Application Date
20240711
Priority Date
20230711

Claims (20)

  1. 1. A cell genetically engineered for producing an oligosaccharide comprising milk-N-trisaccharide (LN 3) or an oligosaccharide mixture comprising an oligosaccharide comprising LN3, wherein the oligosaccharide comprising LN3 is LN3, an oligosaccharide comprising milk-N-tetrasaccharide (LNT) or an oligosaccharide comprising milk-N-neotetrasaccharide (LNnT), characterized in that the cell expresses a transporter comprising an amino acid sequence defined below: (i) Wherein the oligosaccharide comprising LN3 is LN3, an oligosaccharide comprising milk-N-tetraose (LNT), or an oligosaccharide comprising milk-N-neotetraose (LNnT): Said amino acid sequence is selected from SEQ ID NO 01 or 13, or Said amino acid sequence having at least 85.0% sequence identity to the full-length amino acid sequence of SEQ ID NO. 01 or 13, or -A functional fragment having the amino acid sequence of SEQ ID NO. 01 or 13, wherein the functional fragment consists of a number of consecutive amino acid residues from SEQ ID NO. 01 or 13, respectively, and wherein the number is at least 85.0% of the full length of SEQ ID NO. 01 or 13, respectively, or -Said amino acid sequence is a functional fragment of a polypeptide having at least 85.0% sequence identity to the full length amino acid sequence of SEQ ID No. 01 or 13, wherein said functional fragment consists of a number of consecutive amino acid residues from said polypeptide, and wherein said number is at least 85.0% of the full length of said polypeptide; (ii) Wherein the LN 3-containing oligosaccharide is a milk-N-tetraose (LNT) -containing oligosaccharide: -said amino acid sequence is represented by SEQ ID NO. 13, or Said amino acid sequence having at least 85.0% sequence identity to the full-length amino acid sequence of SEQ ID NO. 13, or -A functional fragment having the amino acid sequence of SEQ ID NO. 13, wherein the functional fragment consists of a number of consecutive amino acid residues from SEQ ID NO. 13, and wherein the number is at least 85.0% of the full length of SEQ ID NO. 13, or -Said amino acid sequence is a functional fragment of a polypeptide having at least 85.0% sequence identity to the full length amino acid sequence of SEQ ID No. 13, wherein said functional fragment consists of a number of consecutive amino acid residues from said polypeptide, and wherein said number is at least 85.0% of the full length of said polypeptide; (iii) Wherein the LN 3-containing oligosaccharide is a milk-N-neotetraose (LNnT) -containing oligosaccharide: said amino acid sequence is selected from SEQ ID NO 15 or 16, or Said amino acid sequence having at least 85.0% sequence identity to the full-length amino acid sequence of SEQ ID NO. 15 or 16, or -A functional fragment having the amino acid sequence of SEQ ID NO. 15 or 16, wherein the functional fragment consists of a number of consecutive amino acid residues from SEQ ID NO. 15 or 16, respectively, and wherein the number is at least 85.0% of the full length of SEQ ID NO. 15 or 16, respectively, or -Said amino acid sequence is a functional fragment of a polypeptide having at least 85.0% sequence identity to the full length amino acid sequence of SEQ ID No. 15 or 16, wherein said functional fragment consists of a number of consecutive amino acid residues from said polypeptide, and wherein said number is at least 85.0% of the full length of said polypeptide; (iv) Wherein the LN 3-containing oligosaccharide is LN3: -said amino acid sequence is selected from the group consisting of SEQ ID NO 02, 03, 04, 05 or 06, or -Said amino acid sequence having at least 85.0% sequence identity to the full length amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06, or -Said functional fragment having the amino acid sequence of SEQ ID NO 02, 03, 04, 05 or 06, wherein said functional fragment consists of a number of consecutive amino acid residues from SEQ ID NO 02, 03, 04, 05 or 06, respectively, and wherein said number is at least 85.0% of the full length of SEQ ID NO 02, 03, 04, 05 or 06, respectively, or -Said amino acid sequence is a functional fragment of a polypeptide having at least 85.0% sequence identity to the full length amino acid sequence of SEQ ID No. 02, 03, 04, 05 or 06, wherein said functional fragment consists of a number of consecutive amino acid residues from said polypeptide, and wherein said number is at least 85.0% of the full length of said polypeptide.
  2. 2. The cell of claim 1, wherein the transporter is capable of transporting the LN 3-containing oligosaccharide.
  3. 3. The cell of claim 1 or 2, wherein the transporter is heterologous.
  4. 4. A cell according to any one of claims 1 to 3, wherein the LN 3-containing oligosaccharide is a neutral oligosaccharide.
  5. 5. The cell according to any one of claims 1 to 4, wherein the cell is selected from the list consisting of a microorganism, a plant cell, an animal cell, an insect cell and a protozoan cell.
  6. 6. The cell of any one of claims 1 to 5, wherein the cell is a bacterium.
  7. 7. The cell of any one of claims 1 to 6, wherein the cell expresses a galactoside β -1, 3-N-acetylglucosamine transferase that is involved in the synthesis of said LN 3-containing oligosaccharide.
  8. 8. The cell according to any one of claims 1-7, wherein the cell further expresses one or more glycosyltransferases involved in the synthesis of the LN3 containing oligosaccharide, wherein the one or more glycosyltransferases are selected from the list consisting of galactosyltransferases, fucosyltransferases, N-acetylglucosamine transferases, N-acetylgalactosamine transferases and sialyltransferases.
  9. 9. The cell of any one of claims 1-8, wherein the cell produces a precursor sugar for synthesis of the LN 3-containing oligosaccharide, and/or wherein the cell absorbs a precursor sugar for synthesis of the LN 3-containing oligosaccharide.
  10. 10. The cell of claim 9, wherein the precursor sugar is lactose, optionally wherein the lactose further comprises fucose.
  11. 11. The cell of any one of claims 1 to 10, wherein the cell has the improved production of the LN 3-containing oligosaccharide compared to the cell having the same genetic background but without the transporter.
  12. 12. The cell of claim 11, wherein the improved production comprises: better titer of the sugar (g sugar/liter), and/or Better production rate r (g sugar/liter/hr), and/or Better cell performance index (g sugar/g biomass), and/or Better specific productivity (g sugar/g biomass/hr), and/or Better yield on sucrose (g sugar/g sucrose), and/or Better sucrose uptake/conversion rate (g sucrose/g/hr), and/or Better lactose conversion/consumption rate (g lactose/hr), and/or -An enhanced growth rate of said cells.
  13. 13. The cell of claim 11, wherein the improved production comprises: better titer of the sugar (g sugar/liter), and/or Better production rate r (g sugar/liter/hr), and/or Better cell performance index (g sugar/g biomass), and/or Better specific productivity (g sugar/g biomass/hour).
  14. 14. The cell of any one of claims 1 to 13, wherein the transporter is further: -lack of one or more consecutive amino acids in its transmembrane domain 1 (TM 1) compared to the TM1 of the transporter shown by SEQ ID NO: 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively, or -Comprising one or more non-conservative amino acid substitutions in its transmembrane domain 1 (TM 1), preferably at the following positions compared to TM1 of the transporter shown by SEQ ID NO: 01, 13, 15, 16, 02, 03, 04, 05 or 06, respectively: Positions 24, 28, 31 and/or 32 of the TM1 of the transporter shown by SEQ ID NO. 01, Positions 26, 30, 33 and/or 34 of TM1 of the transporter shown by SEQ ID NO. 13, Positions 16, 20, 23 and/or 24 of the TM1 of the transporter shown by SEQ ID NO. 15, Positions 26, 30, 33 and/or 34 of TM1 of the transporter shown by SEQ ID NO. 16, Positions 22, 26, 29 and/or 30 of the TM1 of the transporter shown by SEQ ID NO. 02, Is represented by SEQ ID NO: 03, position 22 of TM1 of the transporter, Positions 27, 31, 34 and/or 35 of TM1 of the transporter shown by SEQ ID NO: 04, Positions 28, 32, 35 and/or 36 of the TM1 of the transporter shown by SEQ ID NO. 05, Positions 26, 30, 33 and/or 34 of TM1 of the transporter shown by SEQ ID NO. 06; optionally wherein the transporter lacks all amino acids at the N-terminus of its TM1 domain compared to the transporter represented by SEQ ID NO. 01, 13, 02, 03, 04, 05 or 06, respectively.
  15. 15. A method for producing an oligosaccharide comprising milk-N-trisaccharide (LN 3) or an oligosaccharide mixture comprising oligosaccharides comprising LN3, said method comprising the steps of: (a) Culturing the cells according to any one of claims 1-14 in a suitable culture medium and under conditions allowing the production of said LN3 comprising oligosaccharide or said oligosaccharide mixture to form a culture broth; (b) Optionally separating the LN3 containing oligosaccharide from the culture broth or separating any, preferably all, of the oligosaccharides in the mixture from the culture broth.
  16. 16. The method of claim 15, wherein the separating comprises at least one step selected from the list consisting of clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, tangential flow high efficiency filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and ligand exchange chromatography.
  17. 17. The method according to claim 15 or 16, further comprising the step of purifying any, preferably all, of said oligosaccharides comprising LN3 or in said mixture.
  18. 18. The method of claim 17, wherein the purifying comprises at least one of using activated carbon or carbon, using carbon, nanofiltration, ultrafiltration or ion exchange, using alcohols, using aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, or lyophilization.
  19. 19. Use of a cell according to any one of claims 1-14 for the production of an oligosaccharide comprising LN3 or an oligosaccharide mixture comprising oligosaccharides comprising LN 3.
  20. 20. Use of a transporter as defined in any one of claims 1, 2, 3 and 14 for the production of an oligosaccharide comprising LN3 or an oligosaccharide mixture comprising an oligosaccharide comprising LN 3.

Description

Cell production of oligosaccharides comprising lacto-N-trisaccharide (LN 3) Technical Field The invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the invention is in the technical field of culturing genetically engineered cells. The invention describes (i) a method for producing oligosaccharides comprising milk-N-trisaccharide (LN 3) or oligosaccharide mixtures comprising oligosaccharides comprising LN3 by culturing genetically modified cells comprising a transporter, and (ii) genetically modified cells for use in said method. Background To date, oligosaccharides are gaining increasing attention because these diverse molecules exert a range of important biological activities and are widely distributed among all living organisms. An example of such an oligosaccharide is lacto-oligosaccharide (MO) (Usashima T. Et al, 2011, nova Biomedical Books, new York ISBN 978-1-61122-831-1). These oligosaccharides play an important role in a wide variety of normal physiological and pathological processes (e.g., cell transfer, signal transduction, intercellular adhesion, inflammation, and immune response). One example of such sugars is the sugars in milk (Urashima t. Et al, 2011, milk Oligosaccharides, nova Biomedical Books, new York ISBN 978-1-61122-831-1; coppa et al, 2013, ital. J. Pediattr. 2013, 39 (2)), in particular Milk Oligosaccharides (MO), i.e. (oligo) sugars found in milk of animals such as mammals and humans (Urashima et al, 2011; coppa et al, 2013). The structure of the sugar in a large number of milks has been elucidated so far. Most of the milk oligosaccharides found in animals such as mammals and humans contain lactose at the reducing end (Urashima et al, 2011). Other milk oligosaccharides contain N-acetyllactosamine (Gal-. Beta.1, 4-GlcNAc) or milk-N-disaccharide (Gal-. Beta.1, 3-GlcNAc) at the reducing end (Urashima et al, 2011; wrigglesworth et al, 2020, PLoS ONE 15 (12); urashima et al, 2013, biosci. Biotechnol. Biochem 77 (3): p.455-466; wei et al, 2018, sci. Rep.8:4688). Examples hereof are 3-FLN (Gal-. Beta.1, 4- (Fuc-. Alpha.1, 3-) GlcNAc; also known as Lewis x antigen), 3'-SLN (Neu 5 Ac-. Alpha.2, 3-Gal-. Beta.1, 4-GlcNAc), 6' -SLN (Neu 5 Ac-. Alpha.2, 6-Gal-. Beta.1, 4-GlcNAc) (Urashima et al, 2011; wriggle sworth et al, 2020; wei et al, 2018). Such milk, more particularly human milk, has so far been considered as the best food for newborns and infants. It consists of several fractions, of which milk oligosaccharides are the fourth largest fraction. In addition to lactose, human milk and other mammalian milk also contain various structurally diverse oligosaccharides, which are also known as Human Milk Oligosaccharides (HMO) or Mammalian Milk Oligosaccharides (MMO), respectively (Urashima t. Et al, 2011). The importance of MO for nutrition in mammalian and human infants is directly linked to its biological activity, including protection of newborns from pathogens, support of the development of the infant's immune system and cognitive ability. HMOs and MMOs are further known to function as baits to reduce the risk of infection by bacterial and viral pathogens that attach to human cells (by binding to the surface glycoproteins of these cells). In addition, various HMOs and MMOs have anti-inflammatory effects and act as immunomodulators (e.g., reduce the risk of developing food allergies). In summary, these beneficial effects make milk oligosaccharides, especially Mammalian Milk Oligosaccharides (MMO) and Human Milk Oligosaccharides (HMO), attractive components in the nutritional industry for use in the production of infant formulas or as dietary supplements for children and adults. Sugars (e.g., sugars in milk) can be chemically synthesized but are not attractive for several reasons, including stereospecificity issues, product impurities, and high production costs. Accordingly, over the past few years, serious efforts have been made to produce lacto-oligosaccharides in genetically engineered cells such as bacteria (e.g., E.coli (ESCHERICHIA COLI)). Although several milk oligosaccharides have been produced in the art by genetically engineered host cells, there is a constant search for further improvements in the production yield of these oligosaccharides, i.e. achieving higher titers (g sugar/liter culture medium) and/or better cell performance index (g sugar/g biomass). It is therefore an object of the present invention to provide genetically engineered cells for the production of oligosaccharides comprising lacto-N-trisaccharide (LN 3) in an efficient, timely and cost effective manner, and which produce higher amounts of the desired product. Summary of The Invention In a first aspect, the invention provides a cell genetically engineered for the production of an oligosaccharide comprising milk-N-trisaccharide (LN 3) or an oligosaccharide mixture comprising oligosaccharides comprising LN3, wherein the cell expresses, preferably overexpresses, a nov