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Method for preparation of S-adenosylmethionine

A technology for adenosylmethionine and strain production, which is applied in the field of preparing S-adenosylmethionine, and can solve the problems of lack of identification of SAM transporter, biosafety of Escherichia coli and the like

Inactive Publication Date: 2013-11-06
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the cmr(mdfA) gene is a multidrug resistance gene, there may be potential biosafety problems in Escherichia coli transformed by cmr
[0009] Since the 1970s, studies on the transport of SAM in different organisms such as Saccharomyces cerevisiae, rat (liver), Rickettsia praustzii, and African trypanosoma have been studied, but only the hypothesis of the existence of SAM transporters has been put forward, and the No SAM transporters were identified

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  • Method for preparation of S-adenosylmethionine
  • Method for preparation of S-adenosylmethionine
  • Method for preparation of S-adenosylmethionine

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Embodiment 1

[0097] Embodiment 1, gene cloning and plasmid transformation

[0098] The principle of transforming the plasmid is: use the method of genetic engineering to insert pet8, metk and sam2 genes on pET21, and then clone the pet8 gene containing the regulatory sequence into p-metk and p-sam2 clones to realize the co-expression of transporter and synthetase Express. The schematic diagram of each transformed plasmid is shown in Figure 6 .

[0099] 1. Cloning, plasmid construction and expression of pet8 gene

[0100] Since there is no intron in the pet8 gene in the Saccharomyces cerevisiae genome, it can be directly amplified from the genome without RT-PCR. Primers were designed according to the gene sequence (SEQ ID NO: 1) of the pet8 gene in Gene ID: 855729, and the extracted Saccharomycs cerevisiae (Saccharomycs cerevisiae S288c) genomic DNA was used as a template for PCR amplification, and the upstream primer was pet8-1: 5' -CCC AAGCTT ATGAATAC TTTTTTTC-3' (SEQ ID NO: 7); do...

Embodiment 2

[0120] Example 2, the co-cloning expression of metk / sam2 and pet8

[0121] The three plasmids p-pet8, p-metk, and p-sam2 prepared above were further recombined and constructed, and the pet8 gene was placed on p-metk and p-sam2 to achieve co-expression.

[0122] The plasmid design primers of p-pet8 were carried out from the back of XbaI, so that sequences such as RBS were also placed in the multi-cloning site area. PCR amplification was performed using Saccharomyces cerevisiae S288c genomic DNA as a template, and the upstream primer was pt-pet-1:5'-CCC AAGCTT AATAATTTTGTTTAACT-3' (SEQ ID NO: 13); downstream primer is Pt-pet-2: 5'-TTT TTACGCTCTCAT TT-3' (SEQ ID NO: 14); wherein the single underline in the pt-pet-1 primer is the HindIII restriction site, and the double underline in the Pt-pet-2 primer is the XhoI restriction site, so that Connected to the carrier.

[0123] The amplification system is:

[0124]

[0125] The amplification program was: denaturation at 98°C ...

Embodiment 3

[0128] Extraction and purification of SAM in embodiment 3, fermentation product

[0129] Plate the identified E.coli Rosetta strains expressing metk, sam2, pet8, metk-pet8 and sam2-pet8 respectively, and inoculate a single colony in liquid LB (containing 100 μg / mL ampicillin), culture overnight, and 1 The ratio of % (v / v) was inoculated in liquid LB medium, cultivated at 280rpm and 37°C for 3 hours, and when the OD600 reached 0.6, IPTG was added to a final concentration of 0.1mmol / L. After induction for 3 hours, add L-methionine, so that the final concentration of methionine was 0.5g / L, and then samples were taken at 24h and 48h. Centrifuge the obtained bacterial liquid sample at 12000rpm for 10min, discard the precipitate, filter the supernatant with a 0.22um ultrafiltration membrane, and prepare for HPLC detection.

[0130] Referring to the method mentioned in Cai Heng et al. [HPLC—UV method for determination of SAM in fermentation broth; Cai Heng, Zheng Weigang, Wan Honggu...

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Abstract

The invention relates to a method for preparing S-adenosylmethionine. According to the method, a pet8 gene is transferred into an S-adenosylmethionine production strain so that secretory expression of S-adenosylmethionine by the S-adenosylmethionine production strain is obviously promoted; and an S-adenosylmethionine synthetase gene and the pet8 gene are transferred into the S-adenosylmethionine production strain so that a S-adenosylmethionine yield can be further improved. The method solves the technical problem that based on the prior art, an S-adenosylmethionine production strain carries out secretory expression of S-adenosylmethionine difficultly, and promotes separation or purification of a downstream S-adenosylmethionine.

Description

technical field [0001] The invention relates to the field of biochemical industry, more specifically, the invention relates to a method for preparing S-adenosylmethionine. Background technique [0002] S-adenosylmethionine (SAM) is the most important methyl donor in the metabolism of organisms. In addition to transmethylation, it also has transsulfuration and transaminopropyl functions. Participate in the metabolism of homocysteine ​​(SAH), glutathione (GSH) and other important antioxidant active substances and polyamines respectively. Clinically, SAM has high medicinal value and is used in the treatment of diseases such as liver disease, depression and other nervous system diseases, arthritis and fibromyalgia. In the mid-1980s, SAM was first recommended as an antidepressant psychotropic drug in Europe. In 1990, it also passed FDA's randomized double-blind verification in the United States. Due to the constraints of the existing production process, the production cost and...

Claims

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Application Information

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IPC IPC(8): C12P19/40C12N15/63C12N1/21
Inventor 张毅张允斌陆坚峰杨胜利
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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