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Method for rapidly and quantitatively analyzing fermentation metabolites in microbial cell factory

A microbial cell, quantitative analysis technology, applied in the quantitative analysis of microbial cell factory fermentation metabolites, rapid field, can solve the problems of long time, low detection rate, affecting the experimental results, etc., to shorten the detection time, reduce the detection cost, improve the The effect of detection efficiency

Active Publication Date: 2022-04-05
DALIAN UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires three different chromatographic columns, and each column has a different solvent, so the cost is extremely high; in addition, the use of three different chromatographic methods takes a long time and the occupancy rate of detection equipment is high; at the same time, three different chromatographic methods The detection method can easily cause component errors and affect the experimental results
[0007] In summary, the existing detection methods for terpenoids mainly rely on chromatography and mass spectrometry. However, due to the complex composition of the target compounds, on the one hand, the existing detection methods have a low detection rate for the target compounds. On the other hand, although different methods can be used Quantitative analysis of target compounds, but this strategy is costly and time consuming

Method used

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  • Method for rapidly and quantitatively analyzing fermentation metabolites in microbial cell factory
  • Method for rapidly and quantitatively analyzing fermentation metabolites in microbial cell factory
  • Method for rapidly and quantitatively analyzing fermentation metabolites in microbial cell factory

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Construction of recombinant E. coli capable of producing lycopene

[0044] The host bacteria of the present invention is Escherichia Coli BL21 (DE3), and the chromosome is carried by the T7 RNA polymerase gene controlled by the Lacuv5 promoter, and therefore, under IPTG induction, the exogenous gene of T7 promoter driven can be efficiently expressed. The present invention uses plasmid PACYC DUET1 to introduce acetyl-modifyl acetyl transferase gene (ATO), hydroxymethic acid cohase A Synthesis gene (HMGS), hydroxymethylglycate co-enzyme reductase gene (HMGR) into the large intestine Bacillus cells; use plasmid PCDF DUET1 to subtracease gene (IDI), isoprene diphosphate synthase gene (CRTE), octhallyclymened ochanase gene (CRTB), octorate tomato Eulme-desaturase gene (CRTI) is introduced into E. coli cells; a plasmid PRSF DUET1 uses plasmid PRSF DUET1 to de-carboxygenase gene (ERG8), phosphate phosphate gene (ERG8), and methroxylate (ERG8), pyrectyl acid kinase gene (...

Embodiment 2

[0045] Example 2 Metabolic extraction and quantitative detection method during the synthesis of terpenoids

[0046] Picking a single recombinant E. coli in a 5 ml seed medium (LB medium) in 5 ml seed medium (LB medium), in 37 ° C overnight culture, seed culture liquid inoculated with 50 mg / L kanamycin, 30 mg / l chloramphenicol and 100 mg / 青 青 青 青 青 o o o o o o 至 至 600 More than 0.6. 0.3 mM IPTG was added and cultured at 16 ° C for 24 h. Extraction of E. coli fermentation culture solution after IPTG induced by 10 mL was collected by 12000 × g centrifugation for 5 min, and 5 ml of methanol was quenched with 2000 × g centrifugation for 5 minutes, and 1.5 ml extraction solution was added. Ethanol: acetonitrile: water = 2: 2: 1 (v / v), a volume ratio of 1% 2,6-di-tert-butyl pair of cresols) vortex extraction 5 min. The sample was then placed in liquid nitrogen, and the ice was melted for 5 min, and the vortex was extracted from 30s, and repeated 3 times. After the extraction liqui...

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Abstract

The invention discloses a method for rapidly and quantitatively analyzing microbial cell factory fermentation metabolites, and belongs to the technical field of biology. The method comprises the following steps: constructing a mevalonic acid pathway and a lycopene synthesis pathway in an escherichia coli host to obtain escherichia coli capable of biologically synthesizing lycopene, fermenting, centrifugally collecting thalli, quenching, extracting to obtain a sample to be detected, and carrying out qualitative and quantitative analysis through LC-MS (Liquid Chromatography-Mass Spectrometer). The method has the advantages that qualitative and quantitative analysis can be carried out on ten compounds including CoA, Ac-CoA, AcAc-CoA, HMG-CoA, MVA, MVA-5P, IPP, GPP, FPP and GGPP in a sample only through one method, the efficient detection method with high precision and good sensitivity is provided, the previous detection method is greatly simplified, the detection cost is reduced, and the method is suitable for large-scale popularization and application. And multiple target compounds can be extracted and quantitatively detected from a complex matrix.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and more particularly to a rapid, quantitative analysis of the method of fermenting metabolites in microbial cells. Background technique [0002] Terpenoids is a compound of the molecular skeleton with isoprene five-carbon units and their derivatives, which are the largest family of plant secondary metabolism. The terpenoid compound is widely present in nature and is the main component of some plant flavors, resins, pigments, partial animal hormones and vitamins. A variety of terpenoids have important physiological activity and high economic value, such as shrimp, artemisinin, paclitaxel, cannabinoids, etc., these terpenoids have been widely used in food additives, health products, cosmetics, And anti-tumor drugs, etc. [0003] Due to the complex structure of the terpenoids, poor stability, the chemical synthesis steps are cumbersome, low yield, and environmental pollution. Therefore, the main pro...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCY02A50/30
Inventor 康巍马骁薛闯吴又多万慧慧吴芷玥郑丹妮
Owner DALIAN UNIV OF TECH