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Construction of high-yield trans-4-hydroxyproline sucA gene knockout bacteria

A proline and trans technology, applied in the field of high-yield trans-4-hydroxyproline strain construction, can solve the problems of complexity, high cost, and waste

Inactive Publication Date: 2016-03-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Originally, hydroxyproline was obtained by hydrolysis of animal collagen, but the process was complicated, time-consuming, and wasteful
Later, there were also reports on the production of hydroxyproline by microorganisms, but those methods had problems of low yield and high cost, and were difficult to be used in industrial production

Method used

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  • Construction of high-yield trans-4-hydroxyproline sucA gene knockout bacteria
  • Construction of high-yield trans-4-hydroxyproline sucA gene knockout bacteria
  • Construction of high-yield trans-4-hydroxyproline sucA gene knockout bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of vectors containing targeting fragments

[0026] Using the genome of Escherichia coli as a template, the upstream and downstream source arms of the sucA gene were obtained by PCR; using the plasmid pKD4 as a template, the Kan resistance gene was obtained by PCR; then the three were added at a molar ratio of 1:1:1 for fusion PCR to obtain a preliminary targeting fragment containing the upstream and downstream homology arms of the sucA gene and the Kan resistance gene (when designing primers, the 5' end of the upstream homology arm was introduced into NdeI, the 5' end of the downstream homology arm was introduced into XhoI, and the upstream Restriction sites HindIII and BamHI were introduced between the homology arm and the Kan resistance gene. Ligate the fusion fragment with the vector pUCm-T, screen through the Kana resistance plate, carry out NdeI and XhoI double digestion of the correct plasmid, and connect it with the same double digestion p...

Embodiment 2

[0027] Example 2: sucA gene knockout

[0028] ① Prepare the electroporation competence of Escherichia coli BL21(DE3)ΔputA: transfer the plasmid pKD46 (temperature-sensitive plasmid) into the prepared electroporation competence of Escherichia coli BL21(DE3)ΔputA, and then follow the preparation method of electroporation competence , to prepare the corresponding electroporation competent.

[0029] ② Gene knockout: Electrotransfer the targeting fragment into the prepared electroporation competent, and use the Red recombination system to realize the recombination exchange between the targeting fragment and the genome. The revived strains were spread on the Kanna plate for preliminary screening.

[0030] ③Colony PCR verification: Pick a single colony from the plate and save it on another plate at the same time, put the picked colony into sterile water, follow the normal PCR system, use the verification primer as the primer, and run the gel to verify the size of the PCR product . ...

Embodiment 3

[0033] Example 3: Transformation into plasmid pUHVT4, followed by single-factor optimization of fermentation conditions.

[0034] To prepare Escherichia coli BL21(DE3)ΔputAΔsucA competent for transformation, the plasmid pUHVT4 was transformed into competent cells, the transformation system: plasmid 3 μl, KCM 10 μl, water 37 μl, competent 50 μl. After mixing, ice bath for 30 minutes, heat shock at 42°C for 90s, then place on ice for about 5 minutes, add 600 μl of liquid LB medium, recover at 37°C for about 1 hour, and spread ampicillin plate. Afterwards, a single colony was picked and cultured, the plasmid was extracted, and verified by enzyme digestion. Correct bacterial storage will be verified for subsequent fermentation optimization.

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Abstract

The invention provides construction of high-yield trans-4-hydroxyproline sucA gene knockout bacteria and discloses a biosynthesis method of trans-4-hydroxy-L-proline through gene knockout. According to the method, the gene suc A is knocked out on the basis of escherichia coli BL21(DE3) delta put A, the process that alpha-oxoglutarate generates succinic acid or an alpha-oxoglutarate dehydrogenase complex is utilized for catalyzing in the TCA circulation process is disturbed, meanwhile, the trans-4-hydroxy-L-proline hydroxylase gene (hyp) is inserted, and then plasmid pUC19-Ptrp2-hyp-vgb is converted; hydroxylase replaces functions of alpha-ketoglutaricdehydrogenase, 'anaplerosis' is conducted on the TCA circulation process, and it is guaranteed that hydroxyproline is generated while the TCA circulation is conducted. The invention further discloses application of recombinant escherichia coli to production of trans-4- hydroxyproline, it is indicated by the flask shaking fermentation result that the yield of hydroxyproline obtained after mediumoptimization is 1344.1 mg / L and is about 5.53 times that before optimization; compared with original bacteria which are converted into the plasmid pUC19-Ptrp2-hyp-vgb in the same mode, advantages are achieved on the aspect of production of hydroxyproline.

Description

technical field [0001] The invention relates to the construction of a high-yield trans-4-hydroxyproline strain, in simple terms, the method and theory of gene knockout and pathway substitution are used to construct the strain, which belongs to the field of genetic engineering. Background technique [0002] Trans-4-hydroxyproline (trans-4-Hydroxy-L-ProLine), also known as L-hydroxyproline, is the main component of collagen. Hydroxyproline has important application value in many fields, such as medicine, food, biochemistry and beauty industry. For example, in medicine, trans-4-hydroxyproline is a chiral synthon synthesized by many chemicals, such as anti-inflammatory drugs, carbapenems, angiotensin converting enzyme inhibitors, antihypertensive drugs, etc. , due to its multiple physiological functions and relatively unique biological activity, it can be used as a drug for treating soft tissue diseases such as rheumatoid arthritis and damaged connective tissue. The role in fo...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P13/24C12R1/19
Inventor 张震宇林凡孙付保于林
Owner JIANGNAN UNIV
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