A method for increasing the co-production fermentation yield of s-adenosylmethionine and glutathione

A technology of adenosylmethionine and glutathione, which is applied in the field of improving the yield of SAM and GSH co-production and fermentation, can solve the problems of high price, time-consuming, and limited industrial application, and achieves the effect of promoting synthesis and accumulation and improving synthesis rate.

Active Publication Date: 2019-05-24
SUZHOU UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the chemical synthesis method has the defects of complicated process and time-consuming, which limits its industrial application.
Enzymatic synthesis of SAM and GSH has the advantages of high product purity and easy extraction, but the high price of raw material adenosine triphosphate (ATP) has become a limiting factor for enzymatic synthesis of SAM and GSH

Method used

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  • A method for increasing the co-production fermentation yield of s-adenosylmethionine and glutathione
  • A method for increasing the co-production fermentation yield of s-adenosylmethionine and glutathione
  • A method for increasing the co-production fermentation yield of s-adenosylmethionine and glutathione

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment one: Utilize original bacterial strain C.utilis CCTCC M 209298 (WT) shake flask fermentation culture

[0047] The original strain C. utilis CCTCC M 209298 was inoculated into 50 mL of fermentation medium at an inoculum size of 10% (v / v), and cultured on a shaker at 30° C. at 200 rpm for 30 hours.

[0048] Determination of yeast biomass: Yeast biomass was expressed as dry cell weight (DCW). Take 10 mL of fermentation broth, centrifuge at 4000 rpm for 5 min, wash with distilled water for 3 times, collect bacteria, and dry at 70°C until constant weight.

[0049] Extraction and determination of intracellular GSH: Fresh yeast obtained by fermentation culture was washed 3 times with distilled water, treated in 40% ethanol solution at 30°C for 2 hours, centrifuged to take the supernatant as the sample to be tested. The concentration of GSH in the sample was determined by 5,5'-dithiobis(2-nitrobenzoic acid) [DTNB]-glutathione reductase cycle method.

[0050] Extrac...

Embodiment 2

[0053] Embodiment 2: Utilize mutant strain C.utilisΔPor1 (2A) shake flask fermentation culture

[0054] The primer sequences in the examples are shown in List 1 below:

[0055] Table 1: Primer Sequence List

[0056]

[0057] 1. Knockout of the Por1 gene on the mitochondrial membrane of Candida utilis

[0058] (1) The whole genome sequence of C.utilis was retrieved in the National Center for Biotechnology Information (NCBI) database, and the GAP promoter sequence of Saccharomyces cerevisiae was used as a template to find the GAP promoter sequence of C. Primers GAP-for, GAP-rev, using C.utilis CCTCC M 209298 genome as a template to amplify the GAP promoter sequence of C.utilis, the fragment size is about 1000bp;

[0059] (2) After the amplified GAP promoter fragment and the plasmid containing the kan gene fragment are digested, ligated, transformed, and spliced ​​together, PCR amplification is performed using primers kan-for and kan-rev to obtain GAP -kan fragment, the fra...

Embodiment 3

[0070] Example Three: Batch Fermentation Culture Using Original Bacterial Strain C.utilis CCTCC M 209298

[0071] The original bacterial strain C.utilis CCTCC M 209298 was inoculated into a 5L fermenter (BIOTECH-5BGZ, Shanghai Baoxing Bio-Equipment Engineering Co., Ltd.) at an inoculation volume of 10% (v / v), with a liquid volume of 3 L and an inoculum volume of 10% , the temperature is 30°C, the stirring speed is 350rpm, the ventilation rate is 3L / min, the pH is 5.0, and the incubation time is 30 hours.

[0072] Using the same yeast biomass measurement, intracellular GSH extraction and determination, and intracellular SAM extraction and determination methods as in Example 1, after testing, the experimental results using the original strain batch fermentation culture:

[0073] Dry cell weight: 12.67~12.99g / L; SAM production: 184.7~194.1mg / L; GSH production: 205.8~216.9mg / L; combined production of SAM and GSH: 390.5~411.0mg / L; intracellular SAM content: 1.55-1.63%; intracellul...

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Abstract

The invention relates to a method for increasing the coproduced fermentation yield of S-ademetionine and glutathione. According to the method, the gene knockout technology is adopted for knocking out membrane pore protein genes on a mitochondrial membrane of candida utilis, the permeability of the mitochondrial membrane is improved, entry of NADH into mitochondria is promoted, the efficiency of synthesizing ATP through a respiratory chain is improved, and finally the coproduced yield of SAM and GSH in yeast cells is increased. The method provides a new thought for a high yield of similar energy-consuming aerobiotic synthetic compounds in eukaryocytes.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for increasing the co-production fermentation yield of SAM and GSH. Background technique [0002] Both S-adenosylmethionine (SAM) and glutathione (GSH) are important sulfur-containing small molecule active compounds in organisms. [0003] SAM is synthesized enzymatically by the substrate L-methionine and ATP via S-adenosylmethionine synthetase (EC 2.5.1.6). It is an important metabolic intermediate in organisms and participates in more than 40 biochemical reactions in organisms. Transsulfuration and transaminopropyl effects. SAM can increase the levels of GSH, sulfate and taurine in the liver through transsulfurization. It can be used clinically to prevent hepatitis, fatty liver, liver fibrosis, liver cirrhosis and liver cancer. It can also prevent alcohol, drugs and cytokines from affecting the liver. damage. SAM also has anti-inflammatory ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/40C12P21/02C12N15/31C12N15/81C12N1/15C12R1/72
CPCC07K14/40C12P19/40C12P21/02
Inventor 卫功元黎德超王大慧
Owner SUZHOU UNIV
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