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Arthrobacter strain overexpressing hypoxanthine phosphoribosyltransferase gene and its construction method and application

A xanthine phosphoribose and overexpression technology, applied in the fields of genetic engineering and microbial fermentation, can solve problems such as expensive substrates, improved strains, and environmental pollution, and achieve the effects of improving cAMP synthesis ability, increasing production, and reducing costs

Active Publication Date: 2015-09-23
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Arthrobacter strain overexpressing hypoxanthine phosphoribosyltransferase gene and its construction method and application
  • Arthrobacter strain overexpressing hypoxanthine phosphoribosyltransferase gene and its construction method and application
  • Arthrobacter strain overexpressing hypoxanthine phosphoribosyltransferase gene and its construction method and application

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Experimental program
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Effect test

Embodiment 1

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

[0030] Design primers to amplify hgprt by PCR.

[0031] The upstream and downstream primers are:

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

[0033] Downstream primer: GC GTC GAC CTACTCGTAAACGTGCGG (the underline is the SalI restriction site).

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

Embodiment 2

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

[0050] Streak a single colony of Arthrobacter recombinant strain AR-HG on the slant seed medium, and culture it at 30°C for 48-72 hours; scrape a ring and inoculate the bacteria into a 500mL shaker flask containing 50mL liquid seed medium, at 30°C, 200 Cultivate in -250rpm shaking table for 24 hours, prepare seed liquid; With the inoculum amount of 10 (v / v)%, inoculate in the 5L automatic control fermenter that the filling volume is 3L, cultivate in fermentation culture at 30 ℃, control pH7 .0-7.2, ventilation rate 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 ability per unit cell increased by 27.0%, which was 0.606g cAMP / gram 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 in particular relates to an Arthrobacter overexpressing hypoxanthine phosphoribosyltransferase gene and its construction method and application. Background technique [0002] Cyclic adenosine monophosphate (cyclic adenosine-3',5'-monophosphate, referred to as cAMP), is a small molecule with intracellular information transmission function, known as the second messenger, widely exists in the body, and plays a role in glucose metabolism, Various physiological and biochemical processes such as fat metabolism, nucleic acid synthesis, protein synthesis, cell differentiation, carcinogenesis, and reversal 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 system diseases. As an animal feed additive...

Claims

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Application Information

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Patent Type & Authority Patents(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 TECH UNIV