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Recombinant proteus pseudomonadaceae for producing L-xylose and application thereof

A technology of Pseudomonas mutans and recombinant bacteria, applied in the field of bioengineering, can solve the problems of many chemical procedures, many impurities in sugar solution, affecting the extraction process and product quality, and achieve the effect of simple process operation and good fermentation performance

Active Publication Date: 2018-12-21
SHANGHAI LINKCHEM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, xylose is prepared by acid extraction, and sulfuric acid is mostly used for hydrolysis of xylose produced by acid method. However, xylose produced by acid method has the following disadvantages: ① a large amount of sulfuric acid is used in the production process, and sulfuric acid makes the cellulose and hemicellulose in the raw materials and other sugars are hydrolyzed, producing a variety of by-products, which affect the post-extraction process and product quality
②Extraction of xylose by acid method, due to the addition of a large amount of strong acid and strong alkali liquid in the production process, a large amount of production waste liquid is produced in the production process, which has caused huge pollution to the environment
③ Due to the many impurities in the sugar solution of acid hydrolysis, the post-treatment efficiency is low, the chemical procedures are many, the requirements for equipment are high, and the cost of environmental protection is increased
[0004] At present, the production of L-xylose is mainly by the method of chemical synthesis, there are the above-mentioned unfavorable factors
L-xylose can also be converted from L-xylulose, but L-xylulose is a rare sugar with a high price, so it is not suitable as a raw material for the production of L-xylose

Method used

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  • Recombinant proteus pseudomonadaceae for producing L-xylose and application thereof
  • Recombinant proteus pseudomonadaceae for producing L-xylose and application thereof
  • Recombinant proteus pseudomonadaceae for producing L-xylose and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Construction and identification of recombinant plasmid pME6032-2gadh

[0040] Obtain sequences such as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 by chemical synthesis, design primers (2GADH-1, 2GADH-2, 2GADH-3, see Table 1), combine the above 2 The genes of the three subunits of the -ketogluconate dehydrogenase gene (2GADH) were ligated at pME6032 plasmid overnight at 16°C with restriction sites EcoRI, SacI, KpnI, and NcoI, and the ligation product pME6032-gadh was chemically transformed into the large intestine Bacillus JM109 competent cells. Spread the transformation solution on an LB plate containing 50 mg / L tetracycline, extract the plasmid and sequence to verify the constructed recombinant plasmid.

[0041](2) Construction of recombinant strain P.plecoglossicida-2gadh

[0042] The plasmid pME6032-2gadh constructed in (1) was transformed into the starting strain Pseudomonas.plecoglossicida CGMCC 7150 by electroporation, and the positive strain P.plecoglossicid...

Embodiment 2

[0048] The starting strain was Pseudomonas plecoglossicida CGMCC 1.12685, and the rest of the steps were the same as in Example 1, and the positive recombinant strain P.plecoglossicida1-2gadh-25dkg-pdc was screened.

Embodiment 3

[0049] Example 3 Cultivation of recombinant strain P.plecoglossicida-2gadh-25dkg-pdc and L-xylose fermentation

[0050] Seed medium: glucose 15.0g / L, corn steep liquor 4.0g / L, urea 2.0g / L, KH 2 PO 4 2.0g / L, MgS04 7H 2 O 0.5g / L, CaCO 3 1.0g / L, pH 7.0.

[0051] Fermentation medium: glucose 80.0g / L, corn steep liquor 4.0g / L, urea 2.0g / L, KH 2 PO 4 2.0g / L, MgSO 4 ·7H 2 O 0.5g / L, CaCO 3 10.0g / L, pH 6.8.

[0052] Shake flask culture: get appropriate bacterium (gained in embodiment 1) suspension and inoculate in the 500ml shake flask that 50ml seed culture medium is housed, at 30 ℃, cultivate 16-20h under the condition of 220r / min, with 10% inoculum size Transfer to a 500ml shake flask with 50ml of fermentation medium, cultivate to OD at 30°C and 220r / min 650 When it is 0.6, use IPTG to induce, and the final concentration is 0.5mM. The induction temperature is 25°C, fermented for 64 hours, and samples are taken regularly.

[0053] Determination of the output of L-xylo...

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Abstract

The invention discloses recombinant proteus pseudomonadaceae for producing L-xylose and application thereof, and belongs to the technical field of bioengineering. A synthesized 2-ketogluconate reductase gene, a 2,5-diketogluconate reductase gene from corynebaterium ATCC 31090, and a pyruvate decarboxylase gene from saccharomycescerevisiae are successfully expressed in host proteus pseudomonadaceaeby a double-plasmid system; after the obtained gene engineering strain is fermented for 56h on a shaking bottle, the output of L-xylose can reach 16.2g / L, and the conversion rate reaches 20.3%; afterthe obtained gene engineering strain is respectively fermented for 48h and 44h on a 3L fermenting tank and a 15L fermenting tank, the outputs of L-xylose can respectively reach 37.6g / L and 45.8g / L, and the conversion rates of glucose can respectively reach 40.7% and 57.3%. The method has the advantages that the cost of the raw materials is low, the pollution to environment is avoided, the operation is simple, and the important economic benefits and social benefits are realized.

Description

technical field [0001] The invention relates to a recombinant Pseudomonas mutans producing L-xylose and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Xylose is a five-carbon sugar, L-xylose mainly exists in plants and animals. Xylose is a non-calorie sweetener, widely used in food, medicine, chemical industry and other fields. Eating xylose can improve the microbial environment of the human body, improve the body's immunity, and is an ideal sweetener for diabetics. [0003] At present, xylose is prepared by acid extraction, and sulfuric acid is mostly used for hydrolysis of xylose produced by acid method. However, xylose produced by acid method has the following disadvantages: ① a large amount of sulfuric acid is used in the production process, and sulfuric acid makes the cellulose and hemicellulose in the raw materials And other carbohydrates are hydrolyzed to produce a variety of by-products, which affect the po...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12P19/02C12R1/38
CPCC12N9/0006C12N9/88C12N15/78C12P19/02C12Y101/01274C12Y101/99003C12Y401/01001C12N1/20C12N15/52C12N1/205C12R2001/38C12Y101/99004
Inventor 陆茜
Owner SHANGHAI LINKCHEM TECH CO LTD
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