Method for producing uridine diphosphate glucose and special engineering bacteria for method

A technology of uridine diphosphate glucose and uridine monophosphate, which is applied in the field of production of uridine diphosphate glucose, and can solve the problems of harsh reaction conditions, long reaction time, and low yield

Active Publication Date: 2021-01-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical synthesis requires the protection and deprotection of the active groups on the sugar group, the catalyst is expensive, the reaction conditions are harsh, and a large amount of organic solvents are used in the reaction process, which makes the total cost of the chemical synthesis method high and the environment is not friendly
Fermentation often takes a long time and yields are low
There are two main methods for enzymatically synthesizing uridine diphosphate glucose. One is to use uridine triphosphate and glucose-1-phosphate as substrates, which are generated under the action of corresponding pyrophosphorylases. For example, Li Xinliang et al. Phosphoric acid and maltodextrin are used as raw materials, Escherichia coli expressing ur...

Method used

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  • Method for producing uridine diphosphate glucose and special engineering bacteria for method
  • Method for producing uridine diphosphate glucose and special engineering bacteria for method
  • Method for producing uridine diphosphate glucose and special engineering bacteria for method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the construction of recombinant expression vector

[0067] 1. Replace the fragment between the XhoI and SpeI restriction sites of the pBAD-hisB vector with the DNA molecule shown in Sequence 2 to obtain the recombinant expression vector pBAD-GS-AT-EC-1 (sequenced and verified).

[0068] In sequence 2 of the sequence listing, positions 1-1029 from the 5' end encode a polyphosphate kinase derived from Gemmobacter sp.LW-1, positions 1041-3467 encode a sucrose synthase derived from Arabidopsis thaliana, and position 3479 -4204 encodes a uridine monophosphate kinase derived from Escherichia coli.

[0069] The DNA molecule shown in Sequence 2 of the Sequence Listing encodes the protein shown in Sequence 1. In sequence 1 of the sequence listing, positions 1-342 from the N-terminus are polyphosphate kinases derived from Gemmobacter sp.LW-1, positions 343-1150 are sucrose synthases derived from Arabidopsis thaliana, and positions 1151- Position 1391 is uridine mo...

Embodiment 2

[0076] Embodiment 2, the preparation of recombinant engineered bacteria

[0077] 1. The recombinant expression vector pBAD-GS-AT-EC-1 prepared in Example 1 was introduced into Escherichia coli BW25113 to obtain recombinant engineering bacteria TY001.

[0078] 2. The recombinant expression vector pBAD-GS-GM-EC-2 prepared in Example 1 was introduced into Escherichia coli BW25113 to obtain recombinant engineering bacteria TY002.

[0079] 3. The recombinant expression vector pBAD-GS-AC-EC-3 prepared in Example 1 was introduced into Escherichia coli BW25113 to obtain recombinant engineering bacteria TY003.

[0080] 4. Introduce the pBAD-hisB vector into Escherichia coli BW25113 to obtain a control recombinant engineered bacterium.

Embodiment 3

[0081] Embodiment 3, utilize recombinant engineered bacterium to produce uridine diphosphate glucose

[0082] Synthetic principles such as Figure 4 shown.

[0083] 1. Utilization of recombinant engineering bacteria TY001 to produce uridine diphosphate glucose

[0084] 1. The recombinant engineered bacteria TY001 prepared in Example 2 was inoculated into 2YT liquid medium containing 50 μg / ml streptomycin, cultivated at 37° C. and 220 rpm until the OD was 0.8, and arabinose (Shanghai Yuanye Biotechnology Co., Ltd.) was added to the culture system. Co., Ltd., product number: S11032) (the concentration of arabinose in the culture system is 0.2mM), after induction culture at 30°C and 220rpm for 16 hours, the culture system was collected, centrifuged at 4°C and 8000rpm / min for 10min, and the bacterial pellet was collected.

[0085] 2. Use 50mM sodium citrate buffer (pH5.5) to resuspend the bacterial pellet obtained in step 1, ultrasonically (power 195W) for 10min, then add uridin...

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Abstract

The invention discloses a method for producing uridine diphosphate glucose and special engineering bacteria for the method. The method for producing the uridine diphosphate glucose comprises the following steps of by using uridine monophosphate and sucrose as raw materials, producing the uridine diphosphate glucose under the action of the engineering bacteria, wherein the engineering bacteria arerecombinant bacteria for expressing functional protein and are obtained by introducing a gene for encoding the functional protein into starting bacteria, and the functional protein comprises polyphosphokinase, uridine monophosphate kinase and sucrose synthase. The method is of great significance to industrial production of the uridine diphosphate glucose.

Description

technical field [0001] The invention relates to a method for producing uridine diphosphate glucose and special engineering bacteria thereof. Background technique [0002] Uridine diphosphate glucose, referred to as UDP-glucose or UDPG, is a kind of nucleotide sugar widely distributed in microorganisms, animal and plant cells. In organisms, it is used in the biosynthesis of various glycosides, oligosaccharides and polysaccharides as a glucose donor. In addition, as an important intermediate product in the interconversion of monosaccharides or the formation of furfuric acid, it plays a central role in carbohydrate metabolism. [0003] The synthesis methods of uridine diphosphate glucose are mainly chemical synthesis, fermentation and enzymatic conversion. Chemical synthesis requires the protection and deprotection of the active groups on the sugar groups. The catalyst is expensive, the reaction conditions are harsh, and a large amount of organic solvents are used in the reac...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12N9/10C12N9/12C12N1/21C12R1/19
CPCC12P19/305C12N9/1062C12N9/1229C12Y207/04001C12Y207/04014C12Y204/01013
Inventor 胡美荣邱媛媛陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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