Method for synthesizing methyl selenocysteine by using bacillus subtilis

A technology of methylselenocysteine ​​and Bacillus subtilis, which is applied in the field of bioengineering, can solve the problems of complex steps, lengthy process, and shortage, and achieve the effects of reducing production costs, convenient use, and enhancing secretion

Active Publication Date: 2021-11-12
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the preparation methods of SeMCys are mainly chemical synthesis methods, but these chemical synthesis methods have their own shortcomings, such as: complicated steps, or lengthy process, or harsh reaction conditions, or high raw material prices, or involving highly toxic raw materials, etc., causing these Synthetic methods are mostly difficult to put into large-scale practical production, therefore, methylselenocysteine ​​is still very expensive on the market
[0004] However, in the prior art, no bio-fermentation method is used to prepare methylselenocysteine, because the fermentation method has the following advantages: the fermentation cell is easier to prepare, and the cost is low; it is more stable than the isolated enzyme, and is not easily affected by ambient temperature and pH. Influenced by other factors, it is easy to use; no toxic products are produced during the conversion process, and no other by-products are produced, so it is expected to realize the production of industrialized SeMCys with low energy consumption, high efficiency, high purity, and pollution-free
[0005] Although natural microorganisms can enrich organic selenium in bacteria, SeMCys cannot be synthesized due to the lack of related enzymes. At present, only reports have been reported on the expression of selenocysteine ​​methyltransferase from S. Accumulates SeMCys intracellularly, but cannot secrete the product into the medium, thereby limiting its production, and its intracellular production is only 1.14 μg / g dry weight

Method used

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  • Method for synthesizing methyl selenocysteine by using bacillus subtilis
  • Method for synthesizing methyl selenocysteine by using bacillus subtilis
  • Method for synthesizing methyl selenocysteine by using bacillus subtilis

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: Construction of recombinant Bacillus subtilis / pP43NMK-SMT

[0037] (1) Construction of expression vector pP43NMK-SMT

[0038] Chemically synthesize the gene encoding SMT protein (nucleotide sequence shown in SEQ ID NO.1); add pP43NMK homologous sequence (nucleotide sequence shown in SEQ ID NO.13 and The nucleotide sequence is shown in SEQID NO.14) to obtain the SMT fragment; the pP43NMK was digested and recovered by Kpn I and Sma I to obtain the pP43NMK fragment; the SMT fragment and the pP43NMK fragment were assembled using Gibson Assembly Master Mix to obtain the SMT expression vector pP43NMK -SMT.

[0039] Transform the expression vector pP43NMK-SMT into competent cells to obtain transformation products; spread the transformation products in LB culture to obtain transformants, pick the transformants and inoculate them in LB medium for culture, extract plasmids for enzyme digestion verification and sequencing verification , the verification is correct ...

Embodiment 2

[0042] Embodiment 2: Construction of cysteine ​​transporter expression vector

[0043] (1) Extraction of Escherichia coli genomic DNA: Inoculate Escherichia coli strains into LB medium, culture at 200r / min at 37°C for 12h, collect the bacteria by centrifugation, and extract the genomic DNA of Escherichia coli using the bacterial genomic DNA kit of TIANGEN company .

[0044] (2) Design primer sequences to amplify the cysteine ​​transporter gene from the Escherichia coli genomic DNA obtained in the above step (1), the amplification conditions are as follows: pre-denaturation at 94°C for 3min, followed by 30 cycles of 94°C 20s, 55°C for 20s, 72°C for 3min, and finally 72°C for 10min; the specific primers are as follows:

[0045] The gene encoding cysteine ​​transporter Bcr: utilize primer Bcr-F (nucleotide sequence as shown in SEQ ID NO.15) and Bcr-R (nucleotide sequence as shown in SEQ ID NO.16) from step Amplify the Bcr gene in the Escherichia coli genomic DNA obtained in (1)...

Embodiment 3

[0051] Example 3 Construction of recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP1622-cysteine ​​transporter

[0052] The expression vectors pSTOP1622-Bcr, pSTOP1622-YdeD, pSTOP1622-TolC, pSTOP1622-YfiK, pSTOP1622-CydD were transformed into recombinant Bacillus subtilis / pP43NMK-SMT, respectively, to obtain recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP1622-Bcr, Recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP1622-YdeD, Recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP1622-TolC, Recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP1622-YfiK, Recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP1622- CydD; the above transformation products were respectively cultured on kanamycin and tetracycline-resistant plates, and cultured upside down at 37°C for 12 hours until colonies appeared, and a single colony was picked and cultured for preservation, and the genome was extracted for PCR identification, and the recombinants were obtained respectively. Recombinant Bacillus subtilis / pP43NMK-SMT / pSTOP162...

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Abstract

The invention relates to a method for synthesizing methyl selenocysteine by using bacillus subtilis, and belongs to the technical field of biological engineering. According to the invention, recombinant bacillus subtilis which takes bacillus subtilis as a host and expresses a gene for encoding SMT protein and a gene for encoding cysteine transporter protein is constructed, so that extracellular secretion of methyl selenocysteine in the recombinant bacillus subtilis is realized for the first time. By adopting the method for preparing the methyl selenocysteine, the production cost can be greatly reduced, and the method is not easily influenced by factors such as environment temperature and pH and is convenient to use; toxic and harmful products are not generated in the conversion process, and other by-products are not generated; and industrial SeMCys production with low energy consumption, high efficiency, high purity and no pollution is expected to be realized.

Description

technical field [0001] The invention relates to a method for synthesizing methylselenocysteine ​​by using Bacillus subtilis, belonging to the technical field of bioengineering. Background technique [0002] Selenium is an essential trace element for the human body, and it performs physiological functions by forming selenoproteins with selenocysteine ​​as the active center. The bioavailability of selenium varies with the source of selenium and nutritional status, methylselenium is the main active form of low molecular weight selenium metabolites, and methylselenocysteine ​​(SeMCys) acts as a direct supplier of methylated selenium. Body, proved to be one of the most effective anti-cancer selenium compounds, is a more effective selenium source supplement than selenoprotein. [0003] At present, the preparation methods of SeMCys are mainly chemical synthesis methods, but these chemical synthesis methods have their own shortcomings, such as: complicated steps, or lengthy process...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/11C12N15/31C07K14/00C07K14/245C12P13/12C12R1/125
CPCC07K14/00C07K14/245C12P13/12
Inventor 殷娴王凤寰廖永红
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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