Sialyltransferases and their use in producing sialylated oligosaccharides

A technology of sialyltransferase and sialylation, which is applied in the field of providing nutritional preparations, can solve problems such as inability to obtain high purity, hinder extensive scientific evaluation of health beneficial properties, and limit production methods

Pending Publication Date: 2020-05-08
CHR HANSEN HMO GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Little is known about the sialyltransferases that enable the synthesis of sialylated penta- or hexasaccharides such as LST-a, LST-b, or DSLNT, limiting the establishment of production methods for the production of any of these SHMOs
Therefore, these desired oligosaccharides are not available in high purity, hampering a broad scientific assessment of their health beneficial properties

Method used

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  • Sialyltransferases and their use in producing sialylated oligosaccharides
  • Sialyltransferases and their use in producing sialylated oligosaccharides
  • Sialyltransferases and their use in producing sialylated oligosaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1: Development of E. coli Neu5Ac producing strains enabling in vivo screening of sialyltransferases using lactose as acceptor

[0142] Metabolic engineering includes mutagenesis and deletion of specific genes, and genomic integration of heterologous genes, respectively. The genes lacZ and araA were inactivated by mutagenesis using mismatch oligonucleotides as described by Ellis et al. (Proc. Natl. Acad. Sci. USA 98:6742-6746 (2001)).

[0143] Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). To prevent the intracellular degradation of N-acetylneuraminic acid, the genes encoding N-acetylglucosamine-6-phosphate deacetylase (nagA) and glucosamine-6-phosphate deaminase (nagB) and the genes encoding N- Acetylmannosamine kinase (nanK), N-acetylmannosamine-6-phosphate epimerase (nanE), N-acetylneuraminic acid aldolase (nanA) and sialic acid permease (nanT) The entire gene cluster for N-ac...

Embodiment 2

[0155] Example 2: Development of E. coli strains enabling in vivo screening of sialyltransferases using lactose as acceptor but requiring exogenous addition of sialic acid

[0156] E. coli BL21 (DE3) strain #942 was used to establish a screening strain for plasmids encoding sialyltransferases 27 to 100. Therefore, to enable sialic acid uptake and nucleotide activation, the genes nanT and neuA were integrated, respectively. The nanT gene (accession number B21_03035) encoding the major facilitator superfamily transporter of E. coli Neu5Ac was amplified from genomic DNA of E. coli BL21(DE3) and coded for the neuA gene (accession number AF305571) from Campylobacter jejuni sub-optimized and obtained by synthesis. These genes were cloned under the constitutive tetracycline promoter P tet The operon under the control, and use EZ-Tn5 transposase to the resulting expression fragment tet -neuA-nanT-lox66-kan-lox72> (SEQ ID NO: 73) was integrated to obtain screening strain #1730.

[0...

Embodiment 3

[0159] Example 3: Generation of Plasmid Collections Encoding Sialyltransferases

[0160] Gene sequences of characterized or putative sialyltransferases were received from literature and public databases. Since sialyltransferase is generally described as showing higher activity when its signal peptide is missing, the inventors used the online prediction tool SignalP (Petersen et al., Nature Methods, 2011Sep 29; 8(10):785- 6) The corresponding protein sequence was analyzed. Genes were synthesized by GenScript Inc. either as annotated full-length forms or, when signal peptides were predicted, as truncated variants lacking the N-terminal signal peptide (Table 4).

[0161] Sialyltransferases 1 to 26, respectively, were subcloned as operons together with neuA into pDEST14 by SLIC using gene-specific primers (Table 2), resulting in a commonly used type of plasmid: pDEST14-siaT-neuA. The remaining sialyltransferases 27 to 100 were directly subcloned into plasmid pET11a by GenScript ...

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Abstract

Disclosed are methods, genetically engineered cells, sialyltransferases and nucleic acid molecules encoding said sialyltransferases for producing sialylated oligosaccharides as well as the use of saidsialylated oligosaccharides for providing nutritional compositions.

Description

[0001] The present invention relates to sialyltransferases, their use in the production of sialylated oligosaccharides, and to the use of said sialylated oligosaccharides in providing nutritional formulations. Background technique [0002] So far, more than 150 structurally distinct human milk oligosaccharides (HMOs) have been identified. Although HMOs make up only a small amount of total human milk nutrients, their highly beneficial effects on the development of breastfed infants have become apparent over the past few decades. [0003] Up to 20% of the total HMO content in human milk is acidic. Therefore, these HMO molecules have at least one sialic acid moiety. While only 3% of the sialic acid contained in human milk is available in free form, 23% and 74% are bound to (glyco-)proteins and oligosaccharides, respectively. The most common member of the sialic acid family is N-acetylneuraminic acid (Neu5Ac). As part of oligosaccharides, N-acetylneuraminic acid usually leads t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26
CPCC12P19/26A23L33/40C12N9/1085C12N15/70C12Y204/99001C12Y204/99004A23L33/125A23V2002/00C12P19/18
Inventor S·詹尼温D·瓦滕伯格
Owner CHR HANSEN HMO GMBH
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