Method for biosynthesizing D-allose by utilizing D-glucose

A technology of biosynthesis and allose, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., to achieve the effect of increasing yield and yield

Active Publication Date: 2021-03-16
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When glucose and glycerol coexist as a carbon source, wild Escherichia coli consumes glucose preferentially, realizing the co-utilization of glucose and glycerol, and making the bacteria use glucose to efficiently produce D-allose is still a great challenge

Method used

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  • Method for biosynthesizing D-allose by utilizing D-glucose
  • Method for biosynthesizing D-allose by utilizing D-glucose
  • Method for biosynthesizing D-allose by utilizing D-glucose

Examples

Experimental program
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Effect test

Embodiment example 1

[0028] The types of culture medium are as follows

[0029] LB medium:

[0030] Peptone 10g / L

[0031] Yeast powder 5g / L

[0032] Sodium chloride 10g / L

[0033] M9 medium:

[0034]

[0035] The bacterial strain JM109(DE3)-pETDuet-1-dpe-rpiB-gi-pRSFDuet-1-Galp of the synthetic D-allose that will be constructed, (plasmid construction such as figure 1 ) stored at -80°C, take it out and place it at room temperature, take 4 microliters into a 4 ml sterilized LB test tube in an ultra-clean bench, and add ampicillin and kanamycin to one-thousandth of the volume of the test tube culture medium, Cultivate overnight for 14-16h.

[0036]Then, inoculate the above-mentioned bacteria into the shake flask LB medium with one thousandth of ampicillin and kanamycin, the inoculation amount is about one thousandth of the culture volume. , cultured for 48-72 hours, the OD600 of the control group JM109(DE3)-pETDuet-1-pRSFDuet-1 did not change from the experimental group, and the production ...

Embodiment example 2

[0038] On the engineering bacteria chassis cell JM109(DE3), the pathway modification was carried out, and D-fructose was an important intermediate product, and because of its position in the glycolysis pathway, when paying attention to the process of the D-glucose metabolism pathway, it was also Pay attention to the metabolic pathway of D-fructose. Therefore, the order of knockout genes must be FruA, ptsG, and the ccr effect can be removed after knockout, so that the co-utilization of glucose and glycerol can be realized. Then knock out pfka, glk, mak, pfkb. To block the consumption of glucose and fructose. Knockout of genes involved in metabolic pathways. Red homologous recombination is used to knock out FruA and ptsG (the phosphorylation pathway for D-glucose and D-fructose to enter the cell). The purpose of knocking out these two genes is to relieve the ccr effect and improve the efficiency of the non-phosphorylation pathway .

[0039] The primers for gene knockout are ...

Embodiment example 3

[0043] First prepare the bacterial strain carrying the corresponding plasmid, insert 4 microliters of the bacterial strain (JM109DE3 / six-knockout JM109DE3) into an LB test tube, and cultivate overnight at 37°180rmp; amplify, take 20 microliters of seed liquid and insert it into a small tube containing 20 mL of LB medium In shake flasks, culture for two hours, OD is about 0.6-0.8; take out shake flasks and ice bath for 10 minutes to ensure cell viability; aliquot, each tube 1mL into 16 1.5mL centrifuge tubes, centrifuge at 6000rmp 4℃ 4min, discard the supernatant; combine the tubes, suspend the precipitation with 800 microliters of 10% glycerol, 4 tubes are gradually combined into 1 tube, and then centrifuge at 6000rmp 4°C for 4min, discard the supernatant; wash bacteria, use 800 microliters of each tube After suspension with 10% glycerol, centrifuge at 6000rmp for 4 minutes at 4°C, discard the supernatant; repeat the previous step; the last four tubes are suspended with 100 mic...

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Abstract

The invention discloses a method for biosynthesizing D-allose by using D-glucose, belongs to the field of escherichia coli metabolic engineering, and aims to synthesize D-allose by using organisms. Asthe content of D-allose in nature is extremely low and the extraction rate is low, the synthesis of D-arabinose in engineering escherichia coli is a content worthy of research. Three exogenous genesgi, dpe and rpiB are introduced into escherichia coli, and finally D-glucose is isomerized to synthesize D-allose. In order to stabilize the yield, during pathway construction, six genes including FruA, ptsg, pfkB, glk, mak and pfkA are knocked out, and Galp is overexpressed, so that during D-allose synthesis, the co-utilization of various carbohydrates can be realized, glycerol is used for straingrowth, and D-glucose is used for synthesizing D-allose at a higher conversion rate.

Description

technical field [0001] The invention belongs to the field of Escherichia coli metabolic engineering, in particular relates to a method for synthesizing D-allose, in particular to an engineering bacterium for joint production of allose by glucose and glycerol and its construction method and path. Background technique [0002] D-allose is an aldohexose, a rare monosaccharide, a white, odorless crystalline solid, a calorie-free sugar, and has no toxicity. Chemical formula CH 2 OH(CHOH) 4 CHO is isolated from the leaves of an African shrub, but the extraction rate is extremely low, so the artificial synthesis of D-allose becomes more urgent. D-allose, which is the C-3 epimer of glucose, is soluble in water but practically insoluble in methanol. D-allose mainly exists in a cyclic form, consisting of β-D-allo-1.5-pyranose (77.5%), α-D-allo-1.5-pyranose (14%), β-D-allo -1.4-furanose (5%) and α-D-allo-1.4-furanose (3.5%), of which β-D-allo-1.5-pyranose is the main component. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N1/21C12N15/61C12R1/19
CPCC12P19/02C12N9/92C12N9/90C12Y503/01018C12Y503/01006C12Y501/03
Inventor 李灏郑灵洁范立海
Owner BEIJING UNIV OF CHEM TECH
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