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Whole-cell synthesis method of D-psicose by taking glycerol as substrate

A technology of allulose and synthesis method, which is applied in the field of whole-cell synthesis of D-psicose, can solve problems such as difficult large-scale in vitro synthesis, expensive D-glyceraldehyde, troublesome separation and purification, etc., to avoid Purification steps and cofactors, easy separation and purification, and the effect of reducing production costs

Pending Publication Date: 2022-05-03
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the product selectivity of this process is poor, both D-psicose and D-sorbose are present in the product
The two are isomers of each other, and the existence of the by-product D-sorbose has brought great trouble to the separation and purification of the product
In addition, dihydroxyacetone phosphate and D-glyceraldehyde are expensive and unstable, so it is difficult to carry out large-scale in vitro synthesis

Method used

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  • Whole-cell synthesis method of D-psicose by taking glycerol as substrate
  • Whole-cell synthesis method of D-psicose by taking glycerol as substrate
  • Whole-cell synthesis method of D-psicose by taking glycerol as substrate

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Transformation of glycerol into D-psicose by recombinant Escherichia coli whole cell method

[0027] The glycerol kinase encoding gene (glpK, SEQ ID NO: 6), the glycerol 3-phosphate dehydrogenase encoding gene (glpD, SEQ ID NO: 7), the L-fucose-1-phosphate derived from Escherichia coli MG1655 Aldolase encoding gene (FucA, SEQ ID NO: 8) and fructose-1-phosphorylase encoding gene (YqaB, SEQ ID NO: 9) and sugar alcohol oxidase encoding gene (AldO, SEQ ID NO: 10), were cloned between the BamHI and HindIII sites of the vector pET28a (PB) using a one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) respectively (see Table 1 for the primers used). The recipient plasmid was digested with NheI / SalI, the donor plasmid was digested with AvrII / SalI, and assembled by multiple rounds of ePathBricks (specific method reference: Xu, P., et al., 2012, ACS Synthetic Biology, 1(7): 256 -266), the above-mentioned pathway genes were assembled in the form of monocistrons...

Embodiment 2

[0035] Example 2: Preparation of whole-cell catalyst and optimization of D-psicose synthesis conditions

[0036] In order to increase the yield and conversion rate of D-psicose, the present invention further optimizes the conditions for preparing the whole cell catalyst and the reaction conditions for converting glycerol to synthesize D-psicose.

[0037] In the present invention, the preparation process of the whole cell catalyst has been optimized firstly: the concentration range of the inducer IPTG concentration is 0.001-0.5mM, the temperature range of the induced expression of the pathway gene is 15-30°C, and the time range of the induced expression of the pathway gene is 4-30°C. 20h. The results showed that the optimal concentration of inducer IPTG was 0.05mM ( Figure 4 A), the optimum induction temperature is 20°C ( Figure 4 B), the optimal induction time is 12h ( Figure 4 C). The present invention optimizes the reaction conditions for the conversion of glycerin to...

Embodiment 3

[0039] Example 3: Using a fermenter to amplify the reaction process to prepare D-psicose

[0040] In order to verify the feasibility of the D-psicose synthesis method described in the present invention in a small-scale production in a fermenter, on the basis of Example 2, the present invention uses a 5L fermenter to scale up the reaction system. After overnight activation of the recombinant Escherichia coli BL21 / KDABO, transfer it to a fermenter equipped with 3L LB medium at an inoculum size of 2% (v / v), at 37°C, 200rpm, and the condition that the ventilation rate is 4NI / min , cultured to OD 600 = 0.6-0.8. Add IPTG at a final concentration of 0.05mM, and induce for 12h at 20°C, 200rpm, and 4NI / min ventilation to express pathway genes. Under the conditions of 4°C and 5000rpm, centrifuge for 15 minutes to collect the bacteria, wash the bacteria with sterilized ultrapure water to remove the medium, and then resuspend the bacteria in 50mM phosphate buffer with pH 7.5, according ...

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Abstract

The invention provides a whole-cell synthesis method of D-psicose, which is used for converting glycerol into D-psicose by one pot, and relates to the field of biochemical engineering. In order to avoid thermodynamic equilibrium in an isomerase pathway, use of expensive dihydroxyacetone phosphate and D-glyceraldehyde and reduce the production cost of D-psicose, the invention constructs a complete conversion pathway from glycerol to D-psicose. A recombinant strain is used as a whole-cell catalyst, and one-pot conversion of glycerol into D-psicose is realized. Compared with other similar methods, the method disclosed by the invention has the advantages that an intermediate product fructose (an isomeride of D-psicose) is not left in a reaction system, the product is single (only D-psicose is generated, and a by-product D-sorbose is not generated), so that separation and purification are facilitated; compared with an enzyme catalytic reaction system, the method has the advantages that a tedious enzyme purification process is omitted, the production process is simple, the cost is lower, and the method is suitable for large-scale production.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a whole-cell synthesis method of D-psicose using glycerol as a substrate. Background technique [0002] D-Psicose (D-Psicose or D-Allulose) is a rare functional sugar. It is a small molecular weight monosaccharide naturally present in various foods. It was first found in sugarcane and beet molasses, stingray plants and wheat. The sugar content of D-psicose is 70% of that of sucrose, but its calories are only 0.3% of that of sucrose, so it is called non-calorie sugar, and it is an ideal sugar substitute with ultra-low calories. D-psicose is generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. It has enormous physiological benefits, including glycemic suppressive properties, neuroprotective effects, ROS scavenging activity, and anti-obesity effects, among others. D-psicose can also inhibit the accumulation of fat in the abdominal cavity, an...

Claims

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Application Information

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IPC IPC(8): C12P19/02C12N15/70C12N1/21C12N15/53C12N15/54C12N15/60C12R1/19
CPCC12P19/02C12N15/70C12Y207/0103C12Y101/05003C12Y207/01056C12Y401/02017C12N9/0006C12N9/1205C12N9/88C12N9/0004
Inventor 许敬亮吕永坤张辉贾箐熊文龙阿拉牧屈凌波应汉杰王诗元
Owner ZHENGZHOU UNIV
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