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One group of DNA (Deoxyribose Nucleic Acid) molecules, recombinant vector, recombinant ketogulonigenium sp and method for producing 2-keto-L-gulonic acid

A ketoglobulina and DNA molecule technology, applied in the field of genetic engineering, can solve the problems of difficult and incomplete molecular operations, and achieve the effects of promoting growth, increasing acid production level and acid production efficiency

Inactive Publication Date: 2016-08-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although some studies have pointed out that the enzymes in the lipoic acid synthetic metabolic pathway of Bacillus ketoglobulin are incomplete, no definite conclusion has been drawn in this field as to which enzyme is incomplete. There are great difficulties and great challenges, so there are few reports on the construction of lipoic acid modules in Bacillus ketoglobulina

Method used

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  • One group of DNA (Deoxyribose Nucleic Acid) molecules, recombinant vector, recombinant ketogulonigenium sp and method for producing 2-keto-L-gulonic acid
  • One group of DNA (Deoxyribose Nucleic Acid) molecules, recombinant vector, recombinant ketogulonigenium sp and method for producing 2-keto-L-gulonic acid
  • One group of DNA (Deoxyribose Nucleic Acid) molecules, recombinant vector, recombinant ketogulonigenium sp and method for producing 2-keto-L-gulonic acid

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Experimental program
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preparation example Construction

[0047] In the present invention, after the preparation of the recombinant vector is completed, it is transferred into Bacillus ketoglobulina by electroporation under the condition of 2000V, and the specific method is as follows:

[0048] 1. Preparation of K.vulgare (Ketoglobulina) Competent Cells

[0049] K.vulgare glycerol was activated on a solid plate, transferred to 50mL HJ medium, and cultured for 13h until the bacteria reached the late exponential phase; put 40mL of bacteria in a 50mL pre-cooled centrifuge tube, and put it in an ice bath for 15min; freeze at 4°C Centrifuge at 4500rpm for 15min in a centrifuge, discard the supernatant; wash with pre-cooled 10% glycerol, resuspend the cells, centrifuge at 4500rpm for 15min, and remove the supernatant; repeat the previous step; add 30mL of sterilized pre-cooled Cells were resuspended in ultrapure water, centrifuged at 4500rpm for 15min, and the supernatant was discarded; 900μL of pre-cooled 10% glycerol was added to resuspe...

Embodiment 1

[0055] Embodiment 1: the preparation of recombinant ketoglobulina of the present invention

[0056] 1. In vitro amplification of the recombinant vector

[0057] According to the preparation scheme of the DNA molecules and recombinant vectors provided by the present invention, they are synthesized by biotechnology companies, and the vector plasmid map after recombination is shown in figure 1 , wherein the embedded sequence is as shown in SEQ ID NO:3 sequence, that is, KpnI restriction site-16S promoter-SEQ ID NO: sequence shown in 1-5S terminator-HindIII restriction site-6S promoter-SEQ The sequence shown in ID NO: 2-5S terminator-BamHI restriction site.

[0058] The recombinant vector was transformed through the classical heat shock method commonly used in large intestine, and the competent cells used were purchased from Beijing Bomed Technology Development Co., Ltd. BDH5α. After transformation, an appropriate LB resistance plate was selected according to the resistance carr...

Embodiment 2

[0074] Embodiment 2: the shaking flask fermentation test of recombinant ketoglobulina of the present invention

[0075] 1. Test groups

[0076] kv: primitive ketogluconic acid bacteria;

[0077] kv-lxs: the original ketogalactobacillus added with exogenous lipoic acid;

[0078]lip-kv: Recombinant Bacteria ketoclonicus according to the present invention;

[0079] lip-kv-lxs: the recombinant ketoglobulina of the present invention added with exogenous lipoic acid;

[0080] 2. Contrastive test of shake flask fermentation at different time points between kv group and lip-kv group

[0081] The strains of each group were first added to the solid seed medium and cultured at 30°C for 24h for activation, then transferred to the seed medium at the same initial OD and fermented in shake flasks at 30°C and 250r / min, 0h, 3h , 6h, 9h, 12h, 19h, 24h, 30h and other different time points for sampling and analysis.

[0082] Described solid seed culture medium is the seed culture medium afte...

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Abstract

The invention relates to the technical field of genetic engineering, and discloses one group of DNA (Deoxyribose Nucleic Acid) molecules, a recombinant vector, a recombinant ketogulonigenium sp and a method for producing 2-keto-L-gulonic acid. The DNA molecules at least comprise one of nucleotide sequences shown by SEQ ID NO:1 and SEQ ID NO:2. The invention provides the DNA molecules capable of coding a key enzyme in a ketogulonigenium sp lipoic acid metabolic pathway, and the vector is transformed into the ketogulonigenium sp through an embedding way, so that the missing key enzyme can be expressed, endogenous lipoic acid is generated, the growth of bacterial strains is promoted, the acid yield and the acid production efficiency are improved, and the aim of carrying out monoxenie fermentation and acid production without adding exogenous lipoic acid nutritional factors with higher cost can be realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a set of DNA molecules, a recombinant vector, a recombinant ketoglobulina and a method for producing 2-keto-L-gulonic acid. Background technique [0002] Vitamin C is a water-soluble vitamin necessary to maintain the growth and metabolism of the body. It is currently widely used in the fields of medicine, food, feed and cosmetics, and the market is stable. At present, the mainstream production method of vitamin C is the two-step fermentation method, which mainly refers to the process of starting from D-sorbitol and converting it into vitamin C precursor-2-keto-L-gulonic acid through two-step fermentation. In the first step, use Gluconobacter oxydans to convert D-sorbitol into L-sorbose, and in the second step, use a mixed system of ketobacterium and its associated bacteria (mainly Bacillus) to convert L-sorbose For 2-keto-L-gulonic acid. However, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/74C12N1/21C12P7/60C12R1/01
CPCC12N9/20C12P7/60C12Y301/01003
Inventor 元英进潘才惠丁明珠王瑞钊贾楠
Owner TIANJIN UNIV
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