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Method for increasing amount of metabolites in fermentation cells and preparing IDMS standard

A standard product and metabolite technology, applied in the field of microbial fermentation, can solve the problems of hindering the metabolic engineering research process of metabolites, low intracellular metabolite concentration, etc.

Active Publication Date: 2018-10-09
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

traditional preparation 13 C The method of fully labeled standard is obtained by batch culture, but the concentration of intracellular metabolites prepared by this method is usually low
Also, some metabolites of 13 C-labeled corresponding standards or even non-commercialized products hinder the progress of metabolic engineering research on these metabolites

Method used

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  • Method for increasing amount of metabolites in fermentation cells and preparing IDMS standard
  • Method for increasing amount of metabolites in fermentation cells and preparing IDMS standard
  • Method for increasing amount of metabolites in fermentation cells and preparing IDMS standard

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, fermentation and substrate add

[0078] The fermenter uses a 1L tank produced by Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. The volume of the fermentation broth is 0.6L, and the inoculum volume is 1%. The seeds are pre-cultivated at 220rpm and 30°C for 24h, and then 6mL is centrifuged to remove Add 6mL normal saline to redissolve the supernatant, put it into the fermenter, and use OD 600 Test bacteria concentration. The whole process uses NaOH solution to remove CO from the aeration 2, the ventilation rate is 0.6L / min (1vvm), the stirring speed: the initial fermentation group of the substrate supplementation experiment is about 300rpm, and the subsequent 400rpm; the fermentation group without the substrate supplementation experiment is 400rpm, and the fermentation temperature is controlled at 30°C , use ammonia water to control the pH at 5.0, and maintain the tank pressure at 0.05MPa. For the whole fermentation process, the Biostar so...

Embodiment 2

[0082] Embodiment 2, macro data analysis of fermentation process

[0083] Fermentation was carried out by the method of Example 1, and data comparison analysis was performed on the groups without substrate supplementation experiment and the substrate supplementation experiment group. The results are shown in Table 4.

[0084] Table 4

[0085]

[0086]

[0087] The results show that due to the large culture volume, the post-processing time of a single sampling sample is long. In order to minimize the contact time between the sample and cold methanol and prevent a large amount of intracellular metabolites from leaking, add it in 4 times and add it in a single time. 13 The way of C full-standard glucose concentration is 1.5g / L is relatively most suitable. When the dissolved oxygen DO starts to rise and the OUR starts to drop 13 C Add fully labeled glucose, start rapid sampling operation after 5 minutes, each sampling 0.15L. When the oxygen uptake rate (OUR) and carbon d...

Embodiment 3

[0090] Embodiment 3, the acquisition of IDMS standard product

[0091] The method of Example 1 was used to carry out fermentation and substrate supplementation stimulation treatment (supplementation mode 3), and the fermentation broth was extracted to prepare IDMS standard substance. The specific method for preparing IDMS standard items is as follows: figure 1 shown.

[0092] Standards are quantified by known concentration 12 C standard quantitation unknown 13 C metabolite concentration. separate a certain amount of 12 C Phosphate Sugar Standards, 12 C standard organic acids, 12 C nucleotide standards and 12 C Amino Acid Standards Made 4 Kinds of Mixed Standards, 12 The concentration range of standard C is 50-0.05 μmol / L except for amino acids, and the concentration range of amino acids is 200-0.1 μmol / L, and a series of concentration gradients are taken. Finally, the different concentration gradients 12 C mixed standard with 13 C Intracellular metabolites are mixed...

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Abstract

The invention relates to a method for increasing the amount of metabolites in fermentation cells and preparing an IDMS standard. According to the invention, a manner of supplementing a substrate at appropriate time on the basis of batch fermentation is employed for instantaneous supplementation of the substrate in multiple batches, so intracellular metabolites present dynamic response, and the concentrations of a part of the intracellular metabolite are increased; and the fermentation cells are collected at the stage, so a metabolite standard with a <13>C label is efficiently prepared, and theconcentration of the standard is greatly increased.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and more specifically, the invention relates to a method for increasing the amount of metabolites in fermentation cells and preparing IDMS standard products. Background technique [0002] In the field of microbial research and production, in order to efficiently obtain the information required for microbial metabolic engineering, it is necessary to have a comprehensive understanding of the in vivo kinetics of its metabolic pathways and pathway enzymes. However, in the prior art, how to quickly and accurately measure the concentration of intracellular microbial metabolites is a difficult problem, mainly because the concentration of intracellular microbial metabolites is usually low, and the concentration of microbial metabolites in the process of microbial extraction, recovery, processing, etc. The endogenous metabolites will be lost and diluted, further exacerbating the difficulty of this ty...

Claims

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Application Information

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IPC IPC(8): C12N1/16G01N1/28C12R1/84
CPCG01N1/28C12N1/16
Inventor 夏建业舒威李超刘晓云庄英萍
Owner EAST CHINA UNIV OF SCI & TECH
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