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Process for producing adenosylmethionine by metabolic engineering bacteria

A technology of adenosylmethionine and engineering bacteria, applied in the field of high-yield SAM, can solve the problems of difficult control of conditions, high production cost, poor fermentation quality, etc., and achieve the effects of stable yield, low cost and short fermentation period

Inactive Publication Date: 2002-12-18
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

[0009] However, due to the toxicity of methanol to cells and the inhibition of glycerol induced by methanol, the AOX1 expression system must first undergo a 1-2 day glycerol growth period (see Example 6 of this application) in the fermenter, and then add glycerol in a limited amount Make the cells grow to a suitable density, and finally add methanol to induce expression. The whole cycle is as long as 7-12 days. This method of inducing expression, on the one hand, the conditions are difficult to control, and it is easy to infect bacteria; on the other hand, the fermentation quality in the later stage of fermentation Relatively poor, and the production cost is relatively high

Method used

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  • Process for producing adenosylmethionine by metabolic engineering bacteria
  • Process for producing adenosylmethionine by metabolic engineering bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Construction of recombinant expression SAM synthetase 2 plasmid pGAPZαA-SAM2 in methanolic yeast

[0034] 1. Primer design and PCR reaction

[0035] According to the SAM synthetase 2 gene (accession number M23368) derived from S.cerevisiae in GeneBank, two specific primers were designed:

[0036] 5'primer TAT TTCGAA ACC ATG GCCAAGAGCAAAACT

[0037] 3'primer GCGGCCGC GAATTC AGCCTAGCATAAAGAAA

[0038] The primer at the 5' end of the designed gene contains a ribosome binding site, and an Nsp V enzyme cleavage site is introduced at the same time. The primer at the 3' end of the gene contains the sequence near the stop codon TAG and introduces an EcoRI restriction site.

[0039] Extract the total chromosomal DNA of S. cerevisiae genome (Osper et al. 1995, Refined Molecular Biology Experiment Guide, Third Edition), use S. cerevisiae chromosomal DNA as a template, add a certain amount of primers, Taq DNA polymerase and The dNTP mixture was subjected to PCR ...

Embodiment 2

[0044] Example 2 Construction of recombinant cells expressing exogenous SAM synthetase 2

[0045] 1. Preparation of electrotransformed methanol yeast cells

[0046] Inoculate GS115 in 50ml YPD medium, culture at 28~30℃ until OD 600 1.3~1.5, 0~4°C, centrifuge at 4000rpm for 10min, wash the cell pellet with 50ml of frozen sterile water, 25ml of frozen sterile water and 2ml of 1M frozen sorbitol, respectively, each wash at 0~4°C , centrifuge at 4000rpm for 10min to collect the cells, and finally resuspend the centrifuged cells with 100 μl of 1M frozen sorbitol to obtain electroshock competent cells.

[0047] 2. Electrotransformation of competent yeast cells with the recombinant expression plasmid pGAPZαA-SAM2

[0048] About 10 μg of the constructed recombinant expression plasmid pGAPZαA-SAM2 was digested and linearized with Avr II, and the linear DNA was recovered by ethanol precipitation and dissolved in 10 μl of sterile water. At the same time, the empty vector pGAPZαA plasmi...

Embodiment 3

[0051] Example 3 Small amount of rapid extraction of intracellular SAM

[0052] Collect the fermentation broth with known cell concentration by centrifugation, add an equal volume of 20% perchloric acid to extract at 4°C for more than one hour, centrifuge at 12000rpm for 5 minutes, filter the supernatant with a 0.2μm filter membrane, take 20μl from it, and put it on Sample HPLC quantification of SAM.

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Abstract

The present invention provides a method for producing S-adenosyl-L-methionine, SAM by using simple, high-yield and low-cost fermentation, and is characterized by that the exogenous SAM synthetase 2 gene is placed under the control of constitutive GAP promotor, and integrated to methanol yeast to obtain gene engineering bacterium for high-producing SAM. In the simple fermentation culture medium with a certain quantity of externally-added methionine said gene engineering bacterium can be fermented to produce SAM. Said invention also provides a novel strain, the preservative number of said strain is CGMCC No.0746, and its systematic name is Pichia pastoris.

Description

technical field [0001] The present invention relates to the related technology of bioengineering, metabolic engineering and fermentation engineering, specifically, the present invention relates to utilize biotechnology to construct the genetic engineering bacterium of methanol yeast, utilize this genetic engineering bacterium to enhance adenosylmethionine (S-adenosyl-L -methionine, SAM) synthetic metabolic flow to high yield SAM. Background technique [0002] Adenosylmethionine is an important intermediate metabolite in organisms and participates in many biological reactions. SAM is the main methyl donor for the metabolism of organisms. It directly participates in the synthesis of important physiologically active substances such as creatine, adrenaline, epinene and choline, and also participates in the methylation modification of ribose and proteins. Active precursors of sulfur-containing compounds such as glutathione, cysteine, taurine and coenzyme A, which are essential f...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/63C12P19/26
Inventor 袁中一余志良吴星佳李东阳
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI