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Genetically engineered bacterium used for producing 3-hydroxypropionic acid (3-HP) by fermentation, constructing method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and hydroxypropionic acid is applied in the field of genetic engineering to achieve the effects of avoiding insufficient enzyme activity, increasing yield, and strengthening industrial application value

Pending Publication Date: 2017-03-08
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] Although many researchers have constructed numerous genetically engineered bacteria that use glycerol as a substrate to ferment and produce 3-HP through a series of research experiments, which have improved the yield of fermenting and producing 3-HP to a certain extent, but still due to many Due to a variety of known and unknown reasons, the amount of 3-HP fermented with glycerol as a substrate has not reached an ideal state, and the utilization rate of glycerol in the fermentation process also needs to be further improved

Method used

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  • Genetically engineered bacterium used for producing 3-hydroxypropionic acid (3-HP) by fermentation, constructing method and application of genetically engineered bacterium
  • Genetically engineered bacterium used for producing 3-hydroxypropionic acid (3-HP) by fermentation, constructing method and application of genetically engineered bacterium
  • Genetically engineered bacterium used for producing 3-hydroxypropionic acid (3-HP) by fermentation, constructing method and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 Construction of recombinant plasmid pET30a-ALDH-DHAB

[0037] 1. Using the genomic DNA of Klebsiella pneumoniae as a template to amplify the DHAB gene

[0038] Extract the genomic DNA of Klebsiella pneumoniae, use the genomic DNA of Klebsiella pneumoniae as a template, synthesize primers, amplify the DHAB gene sequence, and the primers are as follows:

[0039] P1:ExDHABF:5'-ACGCGGAGCTCATGAAAAGATCAAAACGA-3' (SEQ ID NO.2)

[0040] P2:ExDHABR:5'-CGGCAAGCTTTTAGCTTCCTTTACGCAG-3' (SEQ ID NO.3)

[0041] The upstream primer (P1) introduces the Sac I site, and the downstream primer (P2) introduces the Hind III restriction site. 5 and 4 enzyme cutting site protection bases were introduced at the 5' end respectively.

[0042] The PCR product was analyzed by gel electrophoresis, and the size of the DHAB gene was about 2700bp ( figure 1 ), which is the expected size. After the PCR products were purified, they were sent to Shanghai Sangon Biotechnology Co., Ltd. for...

Embodiment 2

[0067] Induced expression of embodiment 2 transformants

[0068] Pick the single-clonal colony of the recombinant strain from the freshly transformed plate, add 100mL LB medium containing 25μg / mL kanamycin at 37°C, 150rpm, shake and culture for 10-12 hours, until the bacterial solution OD 600 When the value reaches 0.6-0.9, IPTG is added to make the final concentration of IPTG 1.0 mM, induced at 15-25° C. for 15-20 hours, and the engineering bacteria of the present invention are obtained. (Enzyme activity determination is not provided here, the following fermentation experiments can illustrate the characteristics of 3-HP production)

Embodiment 3

[0069] Embodiment 3: engineering bacterium fermentation of the present invention produces the reaction of 3-hydroxypropionic acid

[0070] Add 100mL M9 medium (g.L -1 , glycerin 50, yeast paste 5.0, KH 2 PO 4 7.5, MgSO 4 ·7H 2 O 0.25, (NH 4 ) 2 SO 4 2.0, FeSO 4 ·7H 2 O 0.005, CaCl 20.1, vitamin B120.015, deionized water 1000mL, pH7.0), insert the BL21 (pET30a-ALDH-DHAB) bacterial strain of logarithmic growth phase by 10% inoculum size (same as cultivation method among the embodiment 2), Then cultured on a shaking table at 37° C. and 140 rpm for 60 h. The fermentation broth induced without adding IPTG was used as a control, and the control group was also cultured under the above conditions.

[0071] Samples were taken at regular intervals during the culture, and the 3-HP concentration was determined by HPLC. The yield of 3-HP produced by the genetically engineered bacteria is shown in Table 1 (the values ​​in the table are the average values ​​of three repetitions)....

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Abstract

The invention discloses a genetically engineered bacterium used for producing 3-hydroxypropionic acid (3-HP) by fermentation, a constructing method and application of the genetically engineered bacterium. An acetaldehyde dehydrogenase gene in Saccharomyces cerevisiae and a glycerol dehydratase gene in Klebsiella pneumoniae are connected onto a prokaryotic expression vector pET30a(+) so as to construct a Pet30a-ALDH-DHAB double-gene prokaryotic expression vector, and then the constructed Pet30a-ALDH-DHAB double-gene prokaryotic expression vector is transformed in to an escherichia coli BL21(DE3) competent cell to construct the genetically engineered bacterium used for effectively producing the 3-HP by fermentation. The problems of high energy consumption and sever pollution in chemical industrial synthesis of the 3-HP and the problem of low yield rate in microbial fermentation production of the 3-HP are solved. By utilizing the genetically engineered bacterium to produce the 3-HP by fermentation, a yield of the 3-HP can reach to 8.443g / L, and a production process is simple and convenient for industrialized application.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for fermenting and producing 3-hydroxypropionic acid. The construction method and application thereof belong to the field of genetic engineering. Background technique [0002] 3-hydroxypropionic acid (3-hydroxypropionic acid, 3-HP), also known as β-hydroxypropionic acid, CAS number: 503-66-2. 3-HP is a three-carbon weak organic acid, with There is a hydroxyl group and a carboxyl group to make its molecular properties more active. It is an important chemical intermediate that has emerged in recent years and can be used as a raw material for the production of 1,3-propanediol (PDO), succinic acid, special polyester and acrylic acid , can be used to synthesize coatings, adhesives, water treatment chemicals and personal care products and other chemical products and daily necessities, and has great development value, which makes it attract more and more attention. In 2004, the U.S. Department of En...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0008C12N15/70C12N2800/101C12P7/42C12Y102/0101
Inventor 张传丽王陶董玉玮高明侠李同祥
Owner XUZHOU UNIV OF TECH
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