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Ralstonia engineering bacteria for producing glucose and fermentation production method

A technology of glucose and engineering bacteria, applied in the field of genetic engineering, can solve the problems of missing glycolysis pathway and pentose phosphate pathway, etc., achieve high-efficiency immobilization capacity, efficiently eliminate greenhouse gases, and solve the problem of food shortage

Active Publication Date: 2022-07-29
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of 6-phosphofructokinase and 6-phosphogluconate dehydrogenase, it lacks the complete glycolytic pathway and pentose phosphate pathway

Method used

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  • Ralstonia engineering bacteria for producing glucose and fermentation production method
  • Ralstonia engineering bacteria for producing glucose and fermentation production method
  • Ralstonia engineering bacteria for producing glucose and fermentation production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Glucokinase in Ralstonia H16 glk functional identification

[0038] Ralstonia H16 utilizes fructose, glycerol and CO 2 grow as a carbon source. But the bacteria cannot use glucose. Previous studies have shown that the genome of this bacterium contains all the enzyme genes for glucose metabolism.

[0039] Based on this, the heterologous expression of Zymomonas mobilis ( Zymomonas mobilis ) source of the energy-independent glucose transporter GLF. to not contain glf The pBBR1-MCS2 empty vector of the gene expression cassette was used as a control for fermentation experiments with 10 g / L glucose as the sole carbon source. The result is as figure 1 shown. Ralstonia expressing the glucose transporter has a glucose utilization ability that the control strain does not have. After a lag period of about 2 days, the cell dry weight reached 2.9 g / L after 7 days of fermentation. This result not only indicates that Ralstonia H16 cannot utilize glucose due to ...

Embodiment 2

[0040] Example 2 Construction of Ralstonia H16 ∆glk∆zwf∆ PHB engineered strains

[0041] The mutant Ralstonia chassis cells are mutant strains that knock out the glucokinase gene, the key genes of the ED pathway and the key genes of the PHB synthesis pathway. The glucokinase gene that was knocked out was glk , the key gene of the ED pathway is the gene encoding glucose-6-phosphate dehydrogenase zwf1 (H16_A0316) , zwf2 (H16_B1501) and zwf3 (H16_B2566), the key gene of the PHB synthesis pathway is the gene encoding poly([R]-3 hydroxybutyrate) polymerase phaC1 (H16_A1437), gene encoding acetyl-CoA acetyltransferase phaA (H16_A1438) and acetoacetyl-CoA reductase encoding genes phaB1 (H16_A1439).

[0042] 1. Construction of Ralstonia H16 Δglk strain

[0043] Will glk The gene target knockout vector pK18-glk was introduced into Ralstonia H16 host by combined transfer method. The first round of screening was performed using kanamycin to obtain a host strain in wh...

Embodiment 3

[0070] Embodiment 3 utilizes the Ralstonia engineering strain that produces glucose to ferment glucose with fructose as carbon source

[0071] Ralstonia H16 and glucose-producing Ralstonia H16∆ glk , Ralstonia H16∆ glk ∆ zwf and Ralstonia H16∆ glk ∆ zwf∆ The PHB engineered strains were inoculated into 50 mL of LB liquid medium and rejuvenated at 30°C at 200 rpm for 16 hours.

[0072] Preparation of fermentation medium with fructose as sole carbon source: accurately weigh 3.5 g Na 2 HPO 4 , 1.5 g KH 2 PO 4 , 1.0 g (NH 4 ) 2 SO 4 , 80 mg MgSO 4 ⋅7H 2 O, 1 mg CaSO 4 ⋅2H 2 O, 0.56 mg NiSO 4 ⋅7H 2 O, 0.4 mg ferric citrate and 200 mg NaHCO 3 , 4 g fructose, dilute to 1 L with water as a solvent, and sterilize by high pressure moist heat at 115°C for 30 minutes to obtain a fermentation medium with fructose as the sole carbon source.

[0073] Ralstonia H16 and glucose-producing Ralstonia H16∆ glk , Ralstonia H16∆ glk ∆ zwf and Ralstonia H16∆ glk ∆ zwf∆...

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Abstract

The invention relates to the field of genetic engineering, in particular to a glucose-producing Ralstonia engineering bacterium and a fermentation production method. According to the invention, by knocking out a glucokinase gene (glk) in Ralstonia H16, the accumulation of the product glucose is realized when fructose, glycerol and carbon dioxide (CO2) are used as unique carbon sources. And then, the supply of product precursors glucose-6-phosphoric acid and glucose-1-phosphoric acid in the mutant Ralstonia cells is improved by blocking the diversion of carbon flow by an Enter-Doudoroff (ED) pathway and a phosphopentose poly ([R]-3 hydroxybutyric acid) (PHB) synthesis pathway. Fermentation is carried out by using fructose, glycerol and CO2 as unique carbon sources, and the highest yields of glucose respectively reach 140.8 mg / L, 73.9 mg / L and 253.3 mg / L.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a Ralstonia engineering bacteria producing glucose and a fermentation production method. Background technique [0002] Climate change is causing a new round of global food crisis, greenhouse gases (such as CO 2 ) is the main factor causing extreme weather. Therefore, through technical means to eliminate CO in the atmosphere 2 At the same time, the relief of the food crisis is imminent. As an energy substance, glucose is a monosaccharide synthesized by green plants through photosynthesis, and it is also the basic unit of substances in food and feed materials. At the same time, glucose can be widely added to medicines and condiments. With the development of synthetic biology technology, it has gradually become possible to use metabolic engineering methods for glucose production. [0003] At present, there have been reports on the use of metabolic engineering methods to produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/53C12N15/54C12P19/02C12R1/01
CPCC12N9/1205C12N9/0006C12N9/1029C12N15/52C12N15/74C12P19/02C12Y207/01002C12Y101/01049C12Y101/01036C12Y203/01009
Inventor 王晓璐张杰姚斌罗会颖黄火清苏小运柏映国涂涛王苑王亚茹秦星
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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