Culture method engineering bacterium capable of producing 5-amino acetyl propionic acid in high yield

A kind of technology of aminolevulinic acid and culture method, applied in the field of genetic engineering

Inactive Publication Date: 2012-03-21
CHANGSHA AGREEN BIO TECH LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biological mutagenesis method has complex culture conditions, long cycle and high cost.

Method used

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  • Culture method engineering bacterium capable of producing 5-amino acetyl propionic acid in high yield
  • Culture method engineering bacterium capable of producing 5-amino acetyl propionic acid in high yield
  • Culture method engineering bacterium capable of producing 5-amino acetyl propionic acid in high yield

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]Construction and cultivation of Escherichia coli BL21 (DE3) engineering bacteria producing 5-aminolevulinic acid:

[0032] 1. Extraction of Genomic DNA from Acidophilus Plareriae

[0033] The liquid-cultured Acidophilus paladin was collected by centrifugation, and the DNA was extracted by the CTAB method, and the concentration and integrity of the DNA were detected by agarose. The specific operation is as follows:

[0034] 1). Inoculate the liquid culture medium (1% inoculum size, seal the culture bottle after the culture medium is filled) with acidophilic Rhodobacter paella PSB-1 ​​strain (CCTCC M 206124) and carry out light culture (30°C, light intensity 2000Lx, 7d ). The liquid medium components are: sodium acetate 1640mg / L, MgSO 4 ·7H 2 O 200mg / L, K 2 HPO 4 900mg / L, KH 2 PO 4 600mg / L, CaCl 2 2H 2 O 75mg / L, EDTA 20mg / L, (NH 4 ) 2 SO 4 1320mg / L, FeSO 4 ·7H 2 O 11.8mg / L, trace element solution 1mL / L, pH6.8 (adjusted with NaOH), trace element solution: ...

Embodiment 2

[0069] Construction and cultivation of engineering bacteria producing 5-aminolevulinic acid Escherichia coli M15[PREP4]:

[0070] The basic method is the same as above, the difference is:

[0071] 1). The primers used in the cloning of the ALA synthetase gene are as follows:

[0072] F: 5'-ATTTGAGCTCGGTGCCGTTCTAC-3'

[0073] R: 5'-ACAGTAAGCTTAACTTATTCCGCAGC-3'

[0074] The amplification program was pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 55°C for 60 seconds, extension at 72°C for 60 seconds, 35 cycles, and a final extension of 10 minutes. Detected by 1.0% agarose gel electrophoresis, it showed a single band at 1700bp.

[0075] 2). In the construction of the ALAS gene prokaryotic expression vector, the expression vector was named pQE30-R.A2-hemA. The host bacterium used was M15[PREP4], and the transformed product was cultured on LB plates containing 100 μg / L ampicillin and 50 μg / L kanamycin. The engineering bacterium contai...

Embodiment 3

[0078] Construction and cultivation of engineering bacteria producing 5-aminolevulinic acid Escherichia coli M15[PREP4]:

[0079] The basic method is the same as above, the difference is:

[0080] 1). The primers used in the cloning of the ALA synthetase gene are as follows:

[0081] F: 5'-ATTTGAGCTCGGTGCCGTTCTAC-3'

[0082] R: 5'-AGAATAAGCTTGCAGCGAGGACG-3'

[0083] The amplification program was pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, renaturation at 55°C for 2 minutes, extension at 72°C for 2 minutes, 30 cycles, and a final extension of 10 minutes. The PCR product was detected by 1.0% agarose gel electrophoresis, showing a single band at 2550bp.

[0084] 2). In the construction of the ALAS gene prokaryotic expression vector, the expression vector was named pQE30-R.A3-hemA. The host strain used was M15[PREP4]. Transformation products were spread on LB plates containing 100 μg / L ampicillin and 50 μg / L kanamycin for culture. The enginee...

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Abstract

The present invention relates to a method for cultivating engineering bacterium capable of high yielding 5-amino acetylpropionic acid. The engineering bacterium is colibacillus containing 5-amino acetylpropionic acid synthetase gene of Rhodoblastus acidophilus PSB-1 strain in photosynthetic bacteria. The engineering bacterium is firstly cultured in the culture medium containing peptone, yeast extract and inorganic salt as well as glycine, succinic acid and levulic acid for 2-4 hr and then further cultured in the culture medium with inducing agent added for additional 5-14 hr to reach the maximum output of 5-amino acetylpropionic acid. The present invention has not only high 5-amino acetylpropionic acid output, but also simple technological process, environment friendship and capacity of meeting industrial production requirement.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a method for cultivating engineering bacteria with high yield of 5-aminolevulinic acid. [0002] Background technique [0003] 5-Aminolevulinic acid (5-aminolevulinic acid, hereinafter referred to as ALA), is the common precursor of tetrapyrrole compounds such as porphyrin, (ferrous) heme and vitamin B12, and is a widely present in bacteria and fungi. Non-protein amino acids in living cells of biological organisms such as , animals and plants. As a photodynamic agent, ALA has a wide range of applications in the fields of agrochemical and medical. In the field of agriculture, ALA has no toxic and side effects on plants, is easy to degrade without residue, and has become a promising green and pollution-free seedling strengthening agent, yield increasing agent, stress resistance agent, herbicide, insecticide, color enhancer, and greening agent. and defoliants, etc.; in the field of medicine,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/52C12P13/00C12R1/19
Inventor 张德咏刘勇成飞雪谭新球罗源华何明远张战泓戴建平朱春晖
Owner CHANGSHA AGREEN BIO TECH LTD CO
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