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An engineering strain producing tagatose, its construction method and application

A construction method and technology of engineering strains, applied in the fermentative production of tagatose, in the field of tagatose engineering bacteria, can solve the problems of tagatose production cost increase, not suitable for large-scale production, low yield, etc.

Active Publication Date: 2022-05-10
天津怡和生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production of tagatose is mainly through chemical synthesis and biotransformation. Chemical synthesis requires multiple protection and deprotection steps, resulting in high energy consumption, low yield, and high production costs.
The biotransformation method uses galactose as a raw material, and L-arabinose isomerase catalyzes the synthesis of D-tagatose. This method has a high catalytic synthesis efficiency, but galactose is expensive, and the conversion rate of this reaction is only 50%. The high cost of product separation and the high production cost of tagatose make this biotransformation method unable to be widely used
Relevant patents propose to establish an in vitro catalytic system to convert fructose into tagatose, and increase the conversion rate through phosphorylation and dephosphorylation steps, but expensive ATP is required as a coenzyme, which will increase the production cost of tagatose and is not suitable for large-scale production. Therefore, it is urgent to develop a new method for the synthesis of tagatose with low cost, low pollution and high yield.

Method used

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  • An engineering strain producing tagatose, its construction method and application
  • An engineering strain producing tagatose, its construction method and application
  • An engineering strain producing tagatose, its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Construction of Example 1 Corynebacterium glutamicum recombinant strain Tag1 and Tag2

[0029] 1. Construction of recombinant expression vector pEC-T6PE-T6PP1

[0030] According to the KEGG database, the 6-phosphate tagatose 4-epimerase T6PE gene (SEQ ID NO: 1) derived from Agrobacterium tumefaciens and the 6-phosphate psicose derived from Escherichia coli Phosphorylase T6PP gene (SEQ ID NO: 2), design primer 1, primer 2, primer 3 and primer 4, and obtain corresponding sequence by PCR amplification, gene T6PE1 and expression with restriction endonuclease SacI and SmaI simultaneously The vector pEC-XK99E (Kirchner O and Tauch A.2003, Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum.J.Biotechnol.104:287–299) was digested and ligated to obtain the recombinant plasmid pEC-T6PE1; further The gene T6PP1 and the expression vector pEC-T6PE1 were simultaneously digested and connected with restriction endonucleases SmaI and XbaI to o...

Embodiment 2

[0043] Construction of Example 2 Corynebacterium glutamicum recombinant strain Tag4

[0044] 1. Construction of the integration vector pK18mobsacB-pfk'

[0045] According to the upstream sequence (SEQ ID NO:10) and downstream sequence (SEQ ID NO:11) of the fructose 6-phosphate kinase gene derived from Corynebacterium glutamicum in the KEGG database, design primer 9, primer 10, primer 11 and primer 12, primer 9 and primer 10 were amplified by PCR to obtain the corresponding upstream sequence of the pfk' gene, and primer 9 and primer 10 were amplified by PCR to obtain the corresponding downstream sequence of the pfk" gene; the fusion PCR method was used to obtain the sequence consisting of the upstream and downstream sequences Fusion PCR fragment pfk'-pfk", further use restriction endonuclease EcoRI and HindIII fusion fragment pfk'-pfk" and vector pK18mobsacB( A, Tauch A, Jager W, Kal inowski J, Thierbach G, Puhler A. 1994. SmTag mobilizable multi-purpose cloning vectors deri...

Embodiment 3

[0057] The construction of embodiment 3 Corynebacterium glutamicum recombinant strain Tag5

[0058] 1. Construction of recombinant expression vector pXMJ19-GlK-PGI

[0059] According to the glucokinase GlK gene (SEQ ID NO:4) and the glucose 6-phosphate isomerase PGI gene (SEQ ID NO:5) derived from Corynebacterium glutamicum in the KEGG database, design primer 13, primer 14, primer 15 And primer 16, and take the Corynebacterium glutamicum genome as the gene pgi of template PCR amplification glucokinase gene glk and glucose 6-phosphate isomerase, simultaneously gene pgi and expression vector pXMJ19 with restriction endonuclease HindIII and PstI Carry out enzyme digestion, and connect with T4 ligase to obtain the expression vector pXMJ19-PGI; further use restriction endonuclease PstI and XbaI to carry out enzyme digestion on the gene glk and the expression vector pXMJ19-PGI at the same time, obtain the recombinant expression vector pXMJ19-GlK- PGI. The specific primer sequences...

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Abstract

The invention discloses a construction method and application of an engineering bacterial strain for producing tagatose. The construction method reduces the enzymatic activity of fructose 6-phosphate kinase in cells by regulating glucose intracellular metabolism, and enhances the enzymatic activity of glucokinase and glucose 6-phosphate isomerase so as to increase the content of intracellular fructose 6-phosphate. ‑Phosphotagatose 3‑epimerase and 6‑phosphate tagatose phosphorylase for tagatose synthesis pathway, fructose metabolism pathway consisting of fructose permeation enzyme and fructokinase; obtained a glutamine The recombinant strain of Corynebacterium acid can metabolize glucose, fructose or sucrose to synthesize tagatose. Compared with the currently reported method of bioconverting fructose to synthesize tagatose, it has the advantages of simple operation and easy separation, and is suitable for large-scale production of tagatose. sugar.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering bacterium for producing tagatose, a preparation method of the engineering bacterium and its application in fermenting and producing tagatose. Background technique [0002] Tagatose (D-Tag) is a rare monosaccharide that occurs naturally. It is the ketose form of galactose and the epimer of fructose. The sweetness properties are similar to sucrose, and the calories produced are only one-third of sucrose, so it is called a low-calorie sweetener. Tagatose has excellent nutritional properties such as low calorie value, zero glycemic index, blood sugar passivation, caries-free, prebiotic effect and antioxidant activity. Tagatose has four major functions: low energy, lowering blood sugar, improving intestinal Intestinal flora and anti-caries. The U.S. Food and Drug Administration (FDA) listed D-tagatose as a safe food (GRAS) additive in 2003. The safety of taga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12P19/02C12R1/15
CPCC12N9/1205C12N9/90C12N15/77C12P19/02C12Y207/01144C12Y501/00
Inventor 马延和孙媛霞杨建刚李运杰
Owner 天津怡和生物科技有限责任公司
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