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CN-121825925-B - Beta 1, 4-galactosyltransferase Habeta 4GalT-S233P and application thereof in synthesis of LNnT

CN121825925BCN 121825925 BCN121825925 BCN 121825925BCN-121825925-B

Abstract

The invention discloses beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P and application thereof in synthesis of LNnT, and belongs to the technical field of functional enzymes. The amino acid sequence of the beta 1, 4-galactosyltransferase Habeta 4GalT-S233P is shown in SEQ ID NO. 3. The beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P is applied to the synthesis of LNnT. The beta 1, 4-galactosyltransferase Haβ4GalT-S233P is obtained by modifying beta 1, 4-galactosyltransferase Haβ4GalT, has higher transglycosylation activity, and the conversion rate of the synthesized LNnT can reach 99.80%. The invention lays a foundation for producing LNnT by metabolic engineering reconstruction escherichia coli.

Inventors

  • MAO XIANGCHAO
  • Cao Zhuoning
  • JIANG HONG
  • CHEN RUI
  • Jing Shutong
  • SHI HENGYU

Assignees

  • 中国海洋大学

Dates

Publication Date
20260512
Application Date
20260313

Claims (6)

  1. 1. A beta 1, 4-galactosyltransferase Habeta 4GalT-S233P is characterized in that the amino acid sequence is shown as SEQ ID NO. 3.
  2. 2. The coding gene of the beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P as set forth in claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 4.
  3. 3. Use of the β1, 4-galactosyltransferase Ha β4galt-S233P as claimed in claim 1 for the synthesis of LNnT.
  4. 4. The method of claim 3, wherein the LNnT is produced by reacting beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P with the donor LNT II and the acceptor UDP-Gal.
  5. 5. The application of the beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P in the synthesis of LNnT, which is characterized in that escherichia coli is taken as host bacteria, recombinant engineering bacteria for expressing the beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P are constructed, the recombinant engineering bacteria are cultivated, lactose is added as a substrate, and under the action of the beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P expressed by the recombinant engineering bacteria, UDP-GlcNAc and UDP-Gal which are metabolized and synthesized by the recombinant engineering bacteria are taken as precursor substances to react with lactose to synthesize LNnT.
  6. 6. The use of β 1, 4-galactosyltransferase Ha β 4GalT-S233P for the synthesis of LNnT according to claim 5, wherein the recombinant engineering bacteria expressing β 1, 4-galactosyltransferase Ha β 4GalT-S233P are constructed as follows: (1) Construction of LNnT production base Strain Taking escherichia coli BL21 (DE 3) as host bacteria, knocking out UDP-N-acetyl-2-epimerase gene wecB, purine synthesis repressor gene purR and beta-galactosidase gene lacZ by using a CRISPR-Cas9 gene editing system, integrating sugar transporter gene nagE at wecB gene locus, and integrating PRPP synthetase gene prs at purR gene locus to construct an LNnT production basic strain; The nucleotide sequence of UDP-N-acetyl-2-epimerase gene wecB is shown as SEQ ID NO.5, the nucleotide sequence of purine synthesis repressor gene purR is shown as SEQ ID NO.6, the nucleotide sequence of beta-galactosidase gene lacZ is shown as SEQ ID NO.7, the nucleotide sequence of sugar transporter gene nagE is shown as SEQ ID NO.9, and the nucleotide sequence of PRPP synthetase gene prs is shown as SEQ ID NO. 11; (2) Construction of LNnT-producing Strain Inserting beta 1, 3-N-acetylglucosaminyl transferase gene NmlgtA and lactose permease gene lacY at MCS1 of plasmid pETDueT-1, inserting beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P coding gene at MCS2 of plasmid pETDueT-1 to construct recombinant plasmid; the nucleotide sequence of beta 1, 3-N-acetylglucosaminyl transferase gene NmlgtA is shown as SEQ ID NO.13, the nucleotide sequence of lactose permease gene lacY is shown as SEQ ID NO.15, and the coding gene of beta 1, 4-galactosyltransferase Habeta 4GalT-S233P is shown as SEQ ID NO. 4.

Description

Beta 1, 4-galactosyltransferase Habeta 4GalT-S233P and application thereof in synthesis of LNnT Technical Field The invention relates to beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P and application thereof in synthesis of LNnT, and belongs to the technical field of functional enzymes. Background Breast milk is the best nutrition source for the growth and development of infants, and can meet almost all nutrition requirements for the early growth and development of infants. Breast milk oligosaccharides are structurally complex non-conjugated carbohydrates present in breast milk, being the third largest solid component next to lactose and lipids in breast milk. lactoyl-N-neotetraose (lacto-N-neotetraose, LNnT) is one of the core structures of breast milk oligosaccharide, lactose (Lac) is taken as a reducing end, N-acetylglucosamine and galactose are alternately connected through beta-1, 3 and beta-1, 4 glycosidic bonds to form the tetraose, and the tetraose has the physiological activities of regulating intestinal epithelial barriers, promoting probiotics proliferation, preventing necrotizing enterocolitis and the like, and is a food nutrition enhancer with wide application prospect. Therefore, the large-scale synthesis of LNnT is of great importance. Currently, enzymatic and whole cell synthesis methods are the dominant methods for the synthesis of LNnT. Both synthetic methods are based on beta 1, 4-galactosyltransferase, but the beta 1, 4-galactosyltransferase reported in the prior art has the defects of poor solubility expression, low transglycosylation activity and the like. Therefore, it is of great importance to mine beta 1, 4-galactosyltransferase suitable for industrial use. Disclosure of Invention Aiming at the prior art, the invention provides beta 1, 4-galactosyltransferase Habeta 4GalT-S233P and application thereof in synthesis of LNnT, and belongs to the technical field of functional enzymes. The invention is realized by the following technical scheme: Beta 1, 4-galactosyltransferase Habeta 4GalT-S233P, the amino acid sequence of which is shown in SEQ ID NO. 3. The coding gene of the beta 1, 4-galactosyltransferase Habeta 4GalT-S233P has a nucleotide sequence shown in SEQ ID NO. 4. The beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P is applied to the synthesis of LNnT. The specific application mode is that lactoyl-N-trisaccharide (lacto-N-triose II, LNT II) is taken as a donor, uridine diphosphate galactose (Uridine diphosphate galactose, UDP-galactose, UDP-Gal) is taken as an acceptor, and LNnT is generated by reaction under the action of beta 1, 4-galactosyltransferase Habeta 4 GalT-S233P. The specific application mode can also be that escherichia coli is used as host bacteria to construct recombinant engineering bacteria for expressing beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P, lactose is added as a substrate to culture the recombinant engineering bacteria, and under the action of beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P expressed by the recombinant engineering bacteria, UDP-GlcNAc and UDP-Gal which are metabolized and synthesized by the recombinant engineering bacteria are used as precursor substances to react with lactose to synthesize LNnT. Further, the construction mode of the recombinant engineering bacteria expressing the beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P is as follows: (1) Construction of LNnT production base Strain Taking escherichia coli BL21 (DE 3) as host bacteria, knocking out UDP-N-acetyl-2-epimerase gene wecB, purine synthesis repressor gene purR and beta-galactosidase gene lacZ by using a CRISPR-Cas9 gene editing system, integrating sugar transporter gene nagE at wecB gene locus, and integrating PRPP synthetase gene prs at purR gene locus to construct an LNnT production basic strain; The nucleotide sequence of UDP-N-acetyl-2-epimerase gene wecB is shown as SEQ ID NO.5, the nucleotide sequence of purine synthesis repressor gene purR is shown as SEQ ID NO.6, the nucleotide sequence of beta-galactosidase gene lacZ is shown as SEQ ID NO.7, the nucleotide sequence of sugar transporter gene nagE is shown as SEQ ID NO.9, and the nucleotide sequence of PRPP synthetase gene prs is shown as SEQ ID NO. 11; (2) Construction of LNnT-producing Strain Inserting beta 1, 3-N-acetylglucosaminyl transferase gene NmlgtA and lactose permease gene lacY at MCS1 of plasmid pETDueT-1, inserting beta 1, 4-galactosyltransferase Ha beta 4GalT-S233P coding gene at MCS2 of plasmid pETDueT-1 to construct recombinant plasmid; the nucleotide sequence of beta 1, 3-N-acetylglucosaminyl transferase gene NmlgtA is shown as SEQ ID NO.13, the nucleotide sequence of lactose permease gene lacY is shown as SEQ ID NO.15, and the coding gene of beta 1, 4-galactosyltransferase Habeta 4GalT-S233P is shown as SEQ ID NO. 4. The beta 1, 4-galactosyltransferase Haβ4GalT-S233P is obtained by modifying beta 1, 4-galactosyltransferase Haβ4GalT, has higher transglycosylation activity