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US-12618091-B2 - Production of human milk oligosaccharides in microbial hosts with engineered import/export

US12618091B2US 12618091 B2US12618091 B2US 12618091B2US-12618091-B2

Abstract

The present invention relates to methods for the production of oligosaccharides in genetically modified bacterial host cells, as well as to the genetically modified host cells used in the methods. The genetically modified host cell comprises at least one recombinant glycosyltransferase, and at least one nucleic acid sequence coding for a protein enabling the export of the oligosaccharide.

Inventors

  • Stefan Jennewein
  • Dirk WARTENBERG

Assignees

  • CHR. HANSEN A/S

Dates

Publication Date
20260505
Application Date
20240604
Priority Date
20150912

Claims (7)

  1. 1 . A method for the production of lacto-N-triose II by a genetically modified microbial host cell, comprising providing a genetically modified microbial host cell that comprises: at least one recombinant β-1,3-N-acetylglucosaminyltransferase, wherein the at least one recombinant β-1,3-N-acetylglucosaminyltransferase belongs to the class of lgtA of Neisseria meningitidis , and increased expression or activity of at least one sugar export protein capable of exporting the lacto-N-triose II, wherein the at least one sugar export protein is YjhB from E. coli or ProP from Mannheimia succiniciproducens; cultivating the microbial host cell in a medium under conditions permissive for the production of the lacto-N-triose II, whereby the lacto-N-triose II is exported into the medium at an increased level compared to the unmodified microbial host cell, and obtaining the lacto-N-triose II from the medium.
  2. 2 . The method of claim 1 , wherein, the endogenous β-galactosidase gene and the endogenous glucosamine-6-phosphate deaminase gene of the genetically modified microbial host cell are inactivated or deleted, and wherein said genetically modified microbial host cell comprises a nucleic acid sequence coding for a functional lactose permease protein.
  3. 3 . The method of claim 1 , wherein the genetically modified microbial host cell comprises an increased UDP-N-acetylglucosamine and UDP-galactose or GDP-fucose or CMP-N-acetylneuraminic acid production capability as compared to a genetically unmodified host cell, wherein optionally said increased UDP-N-acetylglucosamine and UDP-galactose production capability comprises the overexpression of one or more genes encoding for proteins comprising the following activities for a: L-glutamine:D-fructose-6-phosphate aminotransferase, N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase, glucose-1-phosphate uridylyltransferase.
  4. 4 . The method of claim 1 , wherein said genetically modified microbial host cell is cultivated in the presence of glucose, sucrose, glycerol or a combination thereof, but not by addition or in the presence of N-acetylglucosamine, galactose or combination thereof.
  5. 5 . A genetically modified microbial host cell for the production of lacto-N-triose II, wherein the microbial host cell comprises: a. at least one recombinant β-1,3-N-acetylglucosaminyltransferase, wherein the at least one recombinant β-1,3-N-acetylglucosaminyltransferase belongs to the class of lgtA of Neisseria meningitidis , and b. increased expression or activity of at least one sugar export protein capable of exporting the lacto-N-triose II, wherein the at least one sugar export protein is YjhB from E. coli or ProP from Mannheimia succiniciproducens.
  6. 6 . The genetically modified microbial host cell of claim 5 , wherein the endogenous β-galactosidase gene and the endogenous glucosamine-6-phosphate deaminase gene in the genetically modified microbial host cell are inactivated or deleted, and wherein said genetically modified microbial host cell comprises a nucleic acid sequence coding for a functional lactose permease protein.
  7. 7 . The genetically modified microbial host cell of claim 5 , wherein the genetically modified microbial host cell comprises an increased UDP-N-acetylglucosamine and UDP-galactose or GDP-fucose or CMP-N-acetylneuraminic acid production capability as compared to a genetically unmodified host cell, wherein optionally said increased UDP-N-acetylglucosamine and UDP-galactose production capability comprises the overexpression of one or more genes encoding for proteins comprising the following activities for a: L-glutamine:D-fructose-6-phosphate aminotransferase, N-acetyl glucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase, glucose-1-phosphate uridylyltransferase.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of U.S. application Ser. No. 17/323,737, published as U.S. patent application publication 2021/0277435 on Sep. 9, 2021, which is a divisional of U.S. application Ser. No. 15/758,653, now U.S. Pat. No. 11,046,985 issued on Jun. 29, 2021, which is a 35 U.S.C. 371 national application of international application no. PCT/EP2016/071420 filed Sep. 12, 2016 and published as International patent application publication WO2017/042385 on Mar. 16, 2017, which claims priority or the benefit under 35 U.S.C. 119 of European application no. 15184968.4 filed Sep. 15, 2015, the contents of which are fully incorporated herein by reference. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing in computer readable form. The contents of the electronic sequence listing created on Jun. 4, 2022, named SQ_ST26.xml and 333 KB in size, is hereby incorporated by reference in its entirety. BACKGROUND Human milk is regarded as the best diet for the development of infants. It is composed of fats, proteins, vitamins, minerals, trace elements and a complex carbohydrate mixture which comprises lactose and approximately 150 structurally diverse oligosaccharides (Human milk oligosaccharides, HMO). Efforts to produce HMO chemically or by biotechnological approaches mainly attracted common attention due to their beneficial impact on the development of the gastrointestinal flora of infants, thus, advocating their use as nutritional additives. Besides these prebiotic properties, many other positive effects of HMO could be observed so far, expanding their field of application. However, extensive scientific studies demand pure single compounds which are hardly achievable. This is especially true for complex free neutral and acidic oligosaccharides for which competitive large-scale production processes are still lacking. (e.g. lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Gluc), lacto-N-neotetraose (Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Gluc), lacto-N-fucopentaose I (Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Gluc) lacto-N-neofucopenaose I (Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Gluc) (Lacto-N-sialylpentaose a (LST-a; Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Gluc)) The metabolic engineering of a microorganism to produce these compounds represents the most promising approach since chemical methods are rather inefficient to produce these molecules at multi-ton scale. Several fermentative approaches were already developed for the structural simpler HMOs such as 2′-fucosyllactose, 3-fucosyllactose or 3′-sialyllactose, using mainly metabolically engineered Escherichia coli strains. However, large-scale quantities are only achievable through boosting the oligosaccharide export out of the bacterial cell, thus, (i) enhancing the productivity and (ii) allowing the recovering of the desired oligosaccharide from the culture broth. The need for solving the export problem seems to enlarge with the size of the produced sugar. Also, with the currently available fermentation processes, upon production of more complex oligosaccharides, the problem of an unwanted export of oligosaccharide precursors from the producing cell occurs, leading to an undesirable mix of product and precursor oligosaccharides in the fermentation medium. Whereas multiple transporter proteins are known to transfer mono- or disaccharides across the membrane, hardly any knowledge exists on the transport of larger oligosaccharides (e.g., trisaccharides and larger oligosaccharides). For example, the genome of the often used fermentation model organism E. coli encodes more than 500 distinct transporter proteins (Busch and Saier, Crit Rev Biochem Mol Biol. 2002; 37(5):287-337). The classification of those membrane transport proteins is quite diverse and subgroups may vary in translocation mechanisms, protein structures or evolutionary origins. Classically energy-driven active transporters perform substrate movement against its concentration or electrochemical gradient, while kinetics and direction of the substrate flow through channels primarily follows such gradients. Depending on the source of energy used for the translocation, pumps can be principally divided into primary active and secondary active transporters, exploiting metabolic energy like ATP or the electrochemical potential, respectively (Davidson and Maloney, Trends Microbiol. 2007 October; 15(10):448-55; Forrest et al, Biochim Biophys Acta. 2011 February; 1807(2):167-88). Although in-depth knowledge was achieved for several membrane proteins permitting energy generation, the import of carbohydrates and the efflux of proteins and antibacterial substances, however, keen insights into mechanistic processes or information on natural or probable substrates were gained only for a minor portion of annotated bacterial transporters so far. The E. coli lactose permease LacY probably represents the most intensively characterized solute transporter