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Production of fucosylated oligosaccharides in bacillus

A technology of Bacillus and fucosylation, which is applied in the field of genetic engineering and can solve problems such as the commercial application of Bacillus has not yet been realized.

Pending Publication Date: 2022-01-21
CHR HANSEN HMO GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0031] Although many patent applications mention Bacillus as a suitable genus for the production of oligosaccharides such as lacto-N-neotetraose, the use of Bacillus for the production of fucosylated oligosaccharides (especially fucoidan commercial application of glycosylated human milk oligosaccharides), presumably because implementing the necessary biosynthetic pathways for HMO production in Bacillus species required extensive efforts in metabolic engineering

Method used

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  • Production of fucosylated oligosaccharides in bacillus

Examples

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Effect test

Embodiment 1

[0179] Example 1: Transformation of Bacillus subtilis

[0180] Bacillus subtilis can be genetically manipulated by a variety of techniques. For transformation of Bacillus subtilis, competent cells were prepared by a modified protocol of the two-step method (Anagnostopoulos, C. and Spizizen, J. (1961) J Bacteriol 81(5):741-746). Inoculate overnight cultures in MG1 medium and shake at 37 °C. MG1 medium is Spizizen's minimal medium supplemented with 0.5% glucose, 5 mM MgSO 4 and 0.02% casamino acids (optionally additionally supplemented with biotin and / or L-tryptophan). The next morning, the culture was diluted 1:20 in fresh MG1 medium and incubated at 37°C for about 6h.

[0181] 1 ml of the culture was diluted in 8 ml of MG2 medium, which differed from MG1 medium in the concentration of casamino acids (0.01% instead of 0.02%). In a shortened protocol, overnight cultures were diluted directly in MG2 medium. After an additional 90 min incubation, 1-ml aliquots of the culture ...

Embodiment 2

[0197] Example 2: Construction of Bacillus subtilis production strains for 2'-fucosyllactose

[0198] Metabolic engineering of sporulation-deficient B. subtilis strains (Table 1) was achieved by integrating the heterologous genes E. coli manC, E. coli manB, and E. coli manA, and simultaneously deleting the endogenous gene lacA by homologous recombination. The Bacillus subtilis gene ganA (yvfN, lacA), located within the galactan operon, encodes a β-galactosidase.

[0199] For the synthesis of GDP-mannose, the open reading frames of manC, manB and manA were operably linked as operators to the Bacillus subtilis constitutive promoter P43 (iGem part repository: Sequence ID: BBa_K143013). The gene manC (Gen Bank accession number: NP_416553.1) encodes GDP:mannose-1-phosphate guanylyltransferase from Escherichia coli. The gene manB (Gen Bank accession number: NP_416552.1) encodes Escherichia coli phosphomannose mutase, and the gene manA (Gen Bank accession number: NP_389084.1) encode...

Embodiment 3

[0201] Example 3: Construction of Bacillus subtilis production strains for 2'-fucosyllactose

[0202] Strain A described in Example 2 was used as a parent strain. Furthermore, for the production of 2'-fucosyllactose from lactose and GDP-mannose, the E. coli genes lacY, gmd, wcaG and wbgL were integrated into the endogenous amyE (amyA) locus of strain A (encoding α-amylase )middle.

[0203] The open reading frame of the gene wbgL (encoding α-1,2-fucosyltransferase) was codon-optimized for expression in Bacillus subtilis, and was prepared synthetically by GenScript. In addition, the opening of genes gmd (Gen Bank accession number: NP_416557.1; encoding GDP-mannose-4,6-dehydratase) and wcaG (Gen Bank accession number: NP_416556.1; encoding GDP-fucose synthase) The reading frame was codon-optimized for expression in Bacillus subtilis (performed by GenScript) as a dual-gene operon placed in the strong constitutive Bacillus subtilis promoter P lepA under the control of Gen...

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Abstract

Disclosed are non-sporulating Bacillus cells for the production of a fucosylated oligosaccharide as well as a method for producing a fucosylated oligosaccharide, wherein said Bacillus cell has been genetically engineered to possess a lactose permease, a GDP-fucose biosynthesis pathway and a fucosyltransferase.

Description

[0001] The present invention relates to the technical field of genetic engineering, in particular to the genetic engineering of Bacillus cells for the production of fucosylated oligosaccharides, and to the use of fucosyl moieties of said genetically engineered Bacillus cells Microbial production of oligosaccharides. Background technique [0002] Human breast milk has optimal nutritional properties for infants. Carbohydrates present in human breast milk represent the main components of human milk, ahead of fat and protein. In addition to lactose as an energy source, human breast milk contains about 5 to 25 g / l of complex sugar molecules called oligosaccharides. These oligosaccharides are only found in significant concentrations in human milk and they are collectively referred to as human milk oligosaccharides (HMOs). [0003] About 200 structurally distinct HMOs have been identified to date. The HMO is based on the disaccharide lactose (consisting of a glucose (Glc) moiety a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N9/10C12N9/12C12N9/04C12N9/88C12N15/54C12N15/53C12N15/60C12P19/18A23L33/21A23C9/20C12R1/125
CPCC12N9/1051C12N9/1205C12N9/88C12N9/0006C12N15/75C12N15/52C12P19/18C12Y204/01069C12Y101/01271C12Y402/01047C12Y207/01A23L33/21A23C9/203A23V2002/00A23V2250/284A23V2200/30A61K31/702C07H3/06A23L33/125C07K14/245C07K14/32Y02A50/30A23L33/135C12N9/2402C12Y302/01023
Inventor S·詹尼温D·瓦滕伯格S·哈伯赛茨
Owner CHR HANSEN HMO GMBH
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