Recombinant strain for producing L-2-aminobutyric acid and preparing method and application thereof

A technology of aminobutyric acid and recombinant bacteria, applied in the biological field, can solve the problems of difficult purification of 2-aminobutyric acid and inability to meet the needs of industrial production, and achieve the effect of improving substrate conversion rate, improving utilization rate, and realizing recycling

Active Publication Date: 2016-11-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent 201210015308.4 synthesizes L-2-aminobutyric acid by overexpressing threonine deaminase and amino acid dehydrogenase on the basis of threonine high-yielding strains. Only 20g / L, still unable to meet the needs of industrial production; enzymatic preparation of L-2-aminobutyric acid is mainly divided into transaminase method, dehydrogenase method, aminoacylase method, aminooxidase method, etc.
But the conversion rate of this method to produce L-2-aminobutyric acid is only 54%, and the output is only 27.8g / L, and the by-product NH is produced in the conversion process 3 , L-alanine and 3-hydroxyl-2-butanone, because the properties of L-2-aminobutyric acid and the by-product alanine are similar, it is difficult to purify the 2-aminobutyric acid prepared by this method

Method used

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  • Recombinant strain for producing L-2-aminobutyric acid and preparing method and application thereof
  • Recombinant strain for producing L-2-aminobutyric acid and preparing method and application thereof
  • Recombinant strain for producing L-2-aminobutyric acid and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Construction of L-2-aminobutyric acid primary engineering bacteria ABA1 and ABA2

[0036] 1. Construction of Threonine Deaminase Expression Plasmid

[0037] Threonine deaminase catalyzes the deamination of threonine to produce 2-ketobutyrate, and overexpression of threonine deaminase promotes the conversion of threonine to 2-ketobutyrate.

[0038] The primers were designed according to the ilvA gene of E.coli K12W3110 in Genbank, the E.coli K12W3110 genome was used as the template, WY1423 and WY1424 were used as primers, and the high-fidelity polymerase KAPA HiFiTM HotStar was used for PCR amplification. The PCR was performed as follows: 94 Denaturation at ℃ for 30 s (seconds), annealing at 54 ℃ for 30 s (seconds), and extension at 72 ℃ for 30 s (seconds) (30 cycles), PCR amplified to obtain a 1560 bp PCR product (sequence 1), the DNA fragment obtained by PCR was subjected to agar After separation and purification by sugar gel electrophoresis, the recovered fragmen...

Embodiment 2

[0060] Example 2, L-2-Aminobutyric acid primary engineering bacteria whole cell catalyzed production of L-2-aminobutyric acid

[0061] L-2-aminobutyric acid primary engineering bacteria ABA1 (BL21 / pET28a-tdcB-ilvE) and ABA2 (BL21 / pET28a-ilvA-ilvE) are used to catalyze the production of L-2-aminobutyric acid.

[0062] The specific seed culture medium is as follows: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L; kanamycin 50mg / L.

[0063] The specific medium for the growth stage of the bacteria is as follows: yeast powder, 10.00g / L; K 2 HPO 4 ·3H 2 O, 0.90g / L; KH 2 PO 4 , 1.14g / L; (NH4) 2 SO 4 , 10.00g / L; MgSO 4 ·7H 2 O, 0.30g / L; trace element (TMS) stock solution, 5.00mL / L.

[0064] Of which TMS storage solution: FeSO 4 ·7H 2 O, 10.000g / L; CaCl 2 , 1.350g / L; ZnSO 4 ·7H 2 O, 2.250g / L; MnSO 4 ·4H 2 O, 0.500g / L; CuSO 4 ·5H 2 O, 1.000g / L; (NH 4 ) 6 Mo 7 O 24 ·4H 2 O, 0.106g / L; Na 2 B 4 O 7 ·10H 2 O, 0.230g / L; CoCl 2 ·6H 2 O, 0.480 g / L; 35% HCl, 10.000 mL / L.

[0065] Buffer: KH 2 PO 4 , 1.36g / ...

Embodiment 3

[0085] Example 3 Construction of L-2-aminobutyric acid engineering bacteria ABA3, ABA4, ABA5

[0086] 1. Construction of multiple transaminase expression plasmids

[0087] Tyrosine aminotransferase TyrB catalyzes the transamination reaction of aromatic amino acids, putrescine aminotransferase PatA catalyzes the transamination reaction of putrescine, and valine pyruvate aminotransferase AvtA catalyzes the formation of alanine from valine and pyruvate. 3-Methyl-2-ketobutyric acid, in order to compare the difference in the catalytic ability of the three aminotransferases to catalyze 2-ketobutyric acid to 2-aminobutyric acid, the expression plasmids and strains containing the three aminotransferases were constructed respectively.

[0088] Using the E.coli K12W3110 genome as a template, using WY1405 and WY1406 as primers, a 1256bp PCR product (SEQ ID No. 8) was amplified; using WY1408 and WY1409 as primers, a 1436bp PCR product was amplified (SEQ ID No. 10). ); Using WY1411 and WY1412 as...

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Abstract

The invention discloses a recombinant strain for producing L-2-aminobutyric acid and a preparing method and application thereof. The recombinant strain is a strain obtained in the mode that threonine dehydratase, transaminase and glutamate dehydrogenase are subjected to overexpression at the same time. Due to the recombinant strain for producing the L-2-aminobutyric acid with threonine serving as a substrate, the yield of the L-2-aminobutyric acid and the substrate conversion ratio are remarkably increased, accumulation of by-products is effectively reduced, and the production technology is simplified.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacteria producing L-2-aminobutyric acid and a preparation method and application thereof. Background technique [0002] L-2-aminobutyric acid is an aliphatic amino acid that inhibits the transmission of human nerve information. It has the functions of reducing blood ammonia, strengthening the activity of glucose phosphatase, and promoting brain cell metabolism. It can be used clinically for the treatment of sequelae of cerebrovascular disease and dental Painless to caries. In addition, L-2-aminobutyric acid is also an important chemical raw material and pharmaceutical intermediate, and has been widely used in the synthesis of the anti-tuberculosis drug ethambutol hydrochloride and the antiepileptic drug levetiracetam. [0003] The production methods of L-2-aminobutyric acid are mainly divided into chemical methods, enzymatic methods and microbial fermentation methods. Chemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/54C12N15/53C12P13/04C12R1/19
Inventor 温廷益温际富梁勇张芸刘树文商秀玲邓爱华刘茜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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