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A recombinant bacterium utilizing dextran to synthesize 3-hydroxypropionic acid and its construction method and application

A technology of hydroxypropionic acid and recombinant bacteria, which is applied in the field of genetic engineering, can solve the problems of limited yield and limited utilization of 3-hydroxypropionic acid substrate, and achieve the effect of increasing yield and substrate utilization

Active Publication Date: 2020-04-24
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the technical bias that researchers usually only consider using common carbon sources such as glycerol and glucose as substrates for microbial transformation, and to solve the problem of limited substrate utilization and limited yield of 3-hydroxypropionic acid synthesized by existing biotransformation methods Problem, the present invention provides a recombinant bacterium that utilizes dextran to synthesize 3-hydroxypropionic acid and its construction method and application

Method used

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  • A recombinant bacterium utilizing dextran to synthesize 3-hydroxypropionic acid and its construction method and application
  • A recombinant bacterium utilizing dextran to synthesize 3-hydroxypropionic acid and its construction method and application

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Embodiment 1

[0049] Construction of embodiment 1 recombinant strain

[0050] 1) Construction of recombinant vector puC18-lgk

[0051] In this example, codon optimization is performed on the gene lgk (the nucleotide number in NCBI is KU377145.1) derived from the levoglucosanokinase of L. starkeyi; the original gene is imported into the website http: / / www.jcat.de / Start.jsp In the process, the commonly used enzyme cutting sites were eliminated, and the optimized gene sequence was obtained after replacing rare codons. The optimized gene sequence was used as a template to synthesize the gene, and its sequence is shown in SEQ ID NO.1. Using the gene obtained by gene synthesis as a template, the gene fragment was obtained by PCR amplification (primers: 5'-CGGGATCCATGCCGATCGCTACCTCTAC-3' and 5'-GCTCTAGATTAAGCCCAGTTGTTGGTGAT-3'); the specific amplification procedure is as follows:

[0052]

[0053] After the PCR, 1% (wt / v) agarose gel electrophoresis was performed, and the target fragment wi...

Embodiment 2

[0065] Embodiment 2 fermentation produces 3-hydroxypropionic acid

[0066] The engineering strain monoclonal that embodiment 1 obtains is activated in LB culture, and the seed liquid after activation is by seed liquid: the ratio of fermentation medium volume ratio 1:130 is inoculated in the 250mL shake flask that contains the fermentation medium of 100mL ( Contains 100μg·mL -1 Kanamycin), cultured with shaking at 35°C and 180rpm. OD 600 When it reaches about 0.8, add 0.1mMIPTG to induce, and continue to cultivate at 37°C. Thereafter, the pH was adjusted to 7 with ammonia water every 12 hours, and the fermentation was terminated 24 hours after the initial induction.

[0067] Take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 min at 4°C, take the supernatant, and detect the product concentration by high-performance liquid chromatography. Using an ultraviolet detector, the 3-HP yield is 0.8 g / L.

Embodiment 3

[0068] Embodiment 3 fermentation produces 3-hydroxypropionic acid

[0069] The engineering strain monoclonal that embodiment 1 obtains is activated in LB culture, and the seed liquid after activation is by seed liquid: the ratio of fermentation medium volume ratio 1:100 is inoculated in the 250mL shake flask that contains the fermentation medium of 100mL ( Contains 100μg·mL -1 Kanamycin), cultured with shaking at 37°C and 180rpm. OD 600 When reaching about 0.6, the temperature was adjusted to 30°C, and 0.01 mM IPTG was added for induction. Thereafter, the pH was adjusted to 7 with ammonia water every 12 hours, and the fermentation was terminated 48 hours after the initial induction.

[0070] Take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 min at 4°C, take the supernatant, and detect the product concentration by high-performance liquid chromatography. Using an ultraviolet detector, the 3-HP yield is 1.1 g / L.

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Abstract

The invention provides recombinant bacteria capable of synthesizing 3-hydracrylic acid by using glucan as well as a construction method and application of the recombinant bacteria, and belongs to the field of gene engineering and fermentation engineering. Klebsiella pneumoniae is host bacteria for expressing a gene lgk capable of encoding levodextran kinase and a gene aldH capable of encoding aldehyde dehydrogenase. The construction method comprises the following steps: obtaining a recombinant carrier puC18-lgk-aldH, converting the recombinant carrier puC18-lgk-aldH into a competent cell of the host Klebsiella pneumoniae, and obtaining the recombinant bacteria. By use of the levodextran kinase gene lgk and the aldehyde dehydrogenase gene aldH which are optimized through a codon, in combination with the whole scheme, the substrate use rate is increased, and the yield is increased.

Description

technical field [0001] The invention relates to a recombinant bacterium for synthesizing 3-hydroxypropionic acid by using dextran, its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] Energy is the basis for the survival and development of modern society. With the development of science and technology, the demand and dependence on energy are also increasing. However, due to the non-renewability of fossil energy, which accounts for 87.7% of the world's primary energy, the fact of energy shortage has become a serious problem before people. The increasing shortage of energy has made the focus of the world's attention on the development and utilization of new energy that can replace fossil energy. Biomass is the only renewable resource that can be converted into liquid fuels. Biomass energy resources are very rich, and the amount of newly generated biomass resources in the world can reach 170 billion ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/22
CPCC12N9/0008C12N9/12C12N15/70C12P7/42C12Y102/01
Inventor 赵广冯新军李申
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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