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Arthrobacterium for overexpression of hypoxanthine phosphoribosyltransferase gene, and building method and application thereof

A technology of xanthine phosphoribosyl and overexpression, which is applied in the fields of genetic engineering and microbial fermentation, can solve the problems of expensive substrates, increased strains, and high production costs, and achieves the effects of improving cAMP synthesis capacity, increasing yield, and increasing yield

Active Publication Date: 2013-09-25
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] Chemical synthesis is currently the main method for industrial production of cAMP at home and abroad, but there are serious defects such as low yield, serious environmental pollution, and high production costs; while enzymatic methods have problems such as poor enzyme stability and expensive substrates, so it is urgent to develop Mild conditions, low cost, environmentally friendly new process to replace
[0004] At present, the modification of strains producing cAMP by fermentation at home and abroad is mainly to screen substrate-tolerant strains or inosinate dehydrogenase-deficient strains through traditional mutagenesis. Increase cAMP production

Method used

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  • Arthrobacterium for overexpression of hypoxanthine phosphoribosyltransferase gene, and building method and application thereof
  • Arthrobacterium for overexpression of hypoxanthine phosphoribosyltransferase gene, and building method and application thereof
  • Arthrobacterium for overexpression of hypoxanthine phosphoribosyltransferase gene, and building method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Construction of genetically engineered strain AR-HG.

[0030] Design primers for PCR amplification of hgprt.

[0031] The upstream and downstream primers are:

[0032] Upstream primer: AA CTGCAG gTTGGTGGATTCAAACGAC (underline is the PstI restriction site);

[0033] Downstream primer: GC GTCGAC CTACTCGTAAACGTGCGG (underlined is the SalI restriction site).

[0034] The PCR product and expression vector pARK were digested with PstI and SalI, respectively. After recovery, hgprt and pARK were ligated with T4 ligase at a ratio of 3:1 to 5:1 at 16°C for 3 hours to construct a recombinant expression vector pARK-HG (Such as figure 1 ). Then, the pARK-HG overexpression plasmid was extracted and concentrated, and the Arthrobacter was transformed by electrotransformation, and recovered at 30°C for 8 hours, coated with a kanamycin resistant plate, and then cultured at 30°C for 36 hours to select transformants. After the transformant is extracted from the plasmid, it is verifi...

Embodiment 2

[0049] Example 2: Fermentation application of Arthrobacter recombinant strain AR-HG.

[0050] A single colony of Arthrobacter recombinant strain AR-HG was streaked to the slant seed medium and cultured at 30°C for 48-72 hours; a loop of bacteria was inoculated into a 500 mL shake flask containing 50 mL liquid seed medium at 30°C, 200 -250rpm shaker for 24 hours to prepare seed liquid; inoculate with 10(v / v)% inoculum into a 5L auto-controlled fermenter with a liquid volume of 3L, cultivate at 30℃ during fermentation culture, and control pH7 .0-7.2, ventilation 8L / min, stirring speed 350rpm, fermentation for 72 hours. The concentration of cAMP in the fermentation broth was detected by HPLC. Compared with the original strain, the yield of recombinant Arthrobacter AR-HG increased by 16.6%, which was 6.04g / L; the cAMP synthesis capacity per cell was increased by 27.0%, which was 0.606g cAMP / gram of cell.

[0051]

[0052]

[0053]

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Abstract

The invention discloses arthrobacterium for overexpression of a hypoxanthine phosphoribosyltransferase gene, and a building method and an application thereof, and clones key enzyme hypoxanthine phosphoribosyltransferase gene (hgprt) of a purine salvage pathway, and rebuilds recombinant expression plasmids by using escherichia coli-arthrobacter shuttle plasmids. The recombinant plasmids are led to the arthrobacterium through an electrotransformation method; a converter is screened by kanamycin resistance, and then genetically engineered bacterium is validated by plasmid PCR (polymerase chain reaction). Thus, the recombinant arthrobacterium AR-HG is built. The yield of the recombinant arthrobacterium AR-HG disclosed by the invention is improved by 16.6% in comparison with that of an original strain; the synthesis ability of unit thallus cAMP (cyclic adenosine-3',5'-monophosphate) is improved by 27.0%; and the arthrobacterium can be directly used for industrial production of the cAMP. Thus, the yield is improved; and the cost is reduced.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and specifically relates to an Arthrobacter overexpressing hypoxanthine phosphoribosyltransferase gene and a construction method and application thereof. Background technique [0002] Cyclic adenosine-3', 5'-monophosphate (cAMP for short) is a small molecule with intracellular information transmission. It is called the second messenger. It exists widely in the body and affects sugar metabolism. Fat metabolism, nucleic acid synthesis, protein synthesis, cell differentiation, carcinogenesis, reversal and many other physiological and biochemical processes play an important regulatory role. Clinically, cAMP can be used to treat cardiovascular diseases, hyperthyroidism, chronic renal insufficiency, nervous system diseases, liver and gallbladder diseases, and respiratory diseases. As an animal feed additive, cAMP has the effect of mimicking growth hormone, can promote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P19/32C12R1/06
Inventor 谢婧婧丁静静应汉杰李楠郭亭朱晨杰陈勇吴菁岚陈晓春
Owner NANJING UNIV OF TECH
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