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Genetic engineering bacteria of gluconobacter oxydans and construction methods thereof

A technology of genetically engineered bacteria and glucose oxidation, applied in the field of genetically engineered bacteria of Gluconobacter oxydans and its construction, can solve the problems of resource and environmental pollution, low vitality, and high raw material prices

Inactive Publication Date: 2013-03-13
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two common problems in the preparation of xylitol by chemical method: (1) the problem of resource and environmental pollution is serious
(2) The price of raw materials is rising, and there is a potential risk of shortage of raw materials
In addition, the xylitol dehydrogenase (Xylitol dehydrogenase, XDH) activity in the bacteria is relatively low, which has become a bottleneck in the process of biological conversion of glucose to xylitol

Method used

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  • Genetic engineering bacteria of gluconobacter oxydans and construction methods thereof
  • Genetic engineering bacteria of gluconobacter oxydans and construction methods thereof
  • Genetic engineering bacteria of gluconobacter oxydans and construction methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Construction of knockout vector for NADP-arabitol dehydrogenase gene.

[0081] (1) Obtain the upstream s-ardh L and downstream s-ardh R and Km genes of the s-ardh gene.

[0082] Two pairs of primers were designed based on the s-ardh sequence of the Gluconobacter oxydans 621H gene published by GenBank:

[0083] s-ardh L-fwd: 5'-TAT GAATTC CCTCTTGAAAACCTATCATAGC-3'EcoR I,

[0084] s-ardh L-rev: 5'-CTGTTTATGTAAGC CTCGAG AAACTTGAAGTCC-3'Xho I,

[0085] s-ardh R-fwd: 5'-AATAAACAAATAG CTCGAGA AAATGGCCGGGAAG-3'Xho I,

[0086] s-ardh R-rev: 5'-AT GAATTC ATGGCGACTGTCGAACTCAAG-3' EcoR I.

[0087] s-ardh L-fwd and s-ardh R-rev primers were added with the same restriction site EcoR I and protective bases at both ends. s-ardh L-rev and s-ardh R-fwd primers add the same restriction enzyme site Xho I at both ends, and the underlined part is the enzyme site.

[0088] By PCR technology, using CGMCC No.2709 genomic DNA as a template, using s-ardh L-fwd and s-ardhL-r...

Embodiment 2

[0103] Embodiment 2: Obtaining of s-ardh gene knockout mutant

[0104] E.coli JM109 containing the knockout mutant vector pSUP202-s-ardh::Km was used as the donor strain for triparental conjugation, and the recipient strain was CGMCC No.2709. The knockout mutant vector entered CGMCCNo.2709 with the help of the helper bacteria pRK2013, and integrated the target gene fragment containing the Km gene into the chromosome of G.oxydans NH-10. Triparental zygotes were screened by Cefotaxime, Km.

[0105] The donor bacteria E.coli JM109 / pSUP202-s-ardh::Km and the helper bacteria JM109 / pRK2013 were inoculated in LB medium with kanamycin and cultured overnight, and the recipient bacteria G.oxydans were inoculated in G- Ara cultured for 15-20 hours, A 660 Conjugation experiments were carried out when the value was 0.6 to 1.0; the bacterial cells were collected according to the volume ratio of the recipient parent bacteria: helper bacteria: donor bacteria = 3:1:3, that is, the recipient ...

Embodiment 3

[0107] Example 3: Construction of xylitol dehydrogenase enhancing vector.

[0108] (1) Obtain the xdh gene

[0109] A pair of specific primers were designed based on the xdh sequence of the Gluconobacter oxydans 621H gene published by GenBank. The primers are as follows:

[0110] xdh-fwd:5'-AGG CTCGAG TCGAAGAAGTTTAAG-3'Xho I,

[0111] xdh-rev: 5'-ATT CTCGAG TCAACCGCCAGCAAT-3'XhoI.

[0112] By PCR technology, with CGMCC No.2709 strain genomic DNA as a template, xdh-fwd and xdh-rev amplified about 800bp xdh sequence ( Figure 9 ). Both ends of xdh-fwd and xdh-rev primers add the same restriction enzyme site Xho I, and the underlined part is the enzyme site.

[0113] xdh gene PCR reaction conditions are: 10×PCR buffer 5μL, Mg 2+ 2 μL, dNTP 5 μL, primers xdh-fwd and xdh-rev 2 μL each, exTaq DNA polymerase 1 μL, genome template 1 μL, add ddH 2 O to a total reaction volume of 50 μL; the PCR reaction was carried out on a PCR machine, and the PCR program was: 94°C pre-denatu...

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Abstract

The invention discloses a genetic engineering bacterium of gluconobacter oxydans, which is the gluconobacter oxydans lacking of an s-ArDH gene. The invention also discloses a genetic engineering bacterium of gluconobacter oxydans, which lacks of an s-ArDH gene and uses the gluconobacter oxydans enhanced by XDH (Xylitol Dehydrogenase) on the position of the s-ArDH gene. The invention also discloses a construction method of two genetic engineering bacteria. The genetic engineering bacteria of the gluconobacter oxydans provided by the invention can block a reverse reaction of xylulose to generate D-arabitol in the process of transforming D-arabitol to prepare xylitol by using a biological method so that the problem of accompanying by-products in the process of manufacturing the xylitol is further radically solved; moreover, an XDH enhanced bacterial strain improves the dehydrogenase activity of the xylitol and increases the conversion rate of the xylitol.

Description

[0001] This patent application is directed at the divisional application whose original application number is 201010190935.2. technical field [0002] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering strain of Gluconobacter oxydans and a construction method thereof. Background technique [0003] Xylitol is a natural five-carbon sugar alcohol and one of the important functional food additives. The sweetness of xylitol is 1.05 times that of sucrose, and its calories are equivalent to that of sucrose. Its metabolism does not require insulin, and it can replace sucrose as a sweetener for diabetics. Xylitol is not fermented by bacteria and is used in chewing gum as a sweetener to maintain oral acid-base balance and prevent dental caries. Studies have shown that xylitol can prevent the combination of bacteria and human cells and prevent respiratory infections; xylitol can also promote the absorption of calcium...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/63C12N15/09C12P19/02C12R1/01
Inventor 徐虹朱宏阳李莎冯小海
Owner NANJING TECH UNIV
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