Construction method and fermentation process of engineering bacteria for producing artemisinin precursor

A technology of artemisinin and construction method, which is applied to the construction of recombinant bacteria for producing artemisinin precursor and the field of fermentation technology, and can solve the problems of high cost, price fluctuation and the like

Inactive Publication Date: 2020-04-17
湖州爱蔻思生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to problems such as high costs and price fluctuations, Sanofi stopped producing artemisinin in 2015 and sold it (Syntheticbiology’s first malaria drug meets market resistance, 2016, Mark Peplow, Naturenews)

Method used

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  • Construction method and fermentation process of engineering bacteria for producing artemisinin precursor
  • Construction method and fermentation process of engineering bacteria for producing artemisinin precursor
  • Construction method and fermentation process of engineering bacteria for producing artemisinin precursor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Strain optimization

[0025] The biosynthetic pathway of this embodiment is as figure 1 As shown, the construction methods of engineered bacteria include global optimization and local optimization, specifically by using predefined regulatory elements (regulatory elements refer to vectors, promoters and 5'untranslated regions) to finely assemble the bacterial library in a high-dimensional, full-combination manner, And combined with analytical chemistry (gas mass spectrometer) to screen the highest yield of microbial strains.

[0026] The flow chart of manufacturing engineered bacteria producing high-value natural products, figure 1 Shown:

[0027] (1) First, establish and characterize the promoter and RBS library through experimental or mathematical methods. The promoter library (T7, TM1-255) was established through experiments, and the specific details can be found in the patent application 2017110721556. The 5’ untranslated region library was established and char...

Embodiment 2

[0033] Example 2: Optimization of basic medium

[0034] Step 1) Prepare 5L of chemically determined medium, including 10g / L glucose, 2g / L(NH 4 ) 2 SO 4 , 4.2g / LKH 2 PO 4 And 11.24g / LK 2 HPO 4 , 1.7g / L citric acid, 0.5g / L MgSO 4 And 10mL / L trace element solution. Among them, the trace element solution contains 0.25g / L CoCl 2 ·6H 2 O, 1.5g / L MnSO 4 ·4H 2 O, 0.15g / L CuSO 4 ·2H 2 O, 0.3g / LH 3 BO 3 , 0.25g / L Na 2 MoO 4 ·2H 2 O,0.8g / L Zn(CH 3 COO) 2 , 5g / L iron (III) citrate and 0.84g / L EDTA, the pH of the trace element solution is 8.0.

[0035] Step 2) Sterilize the culture medium at 121°C for 15-20 minutes. (Note, the glucose solution should be sterilized separately and then mixed with other ingredients).

[0036] Step 3) Next, the strain 323-2 is pre-cultured in the chemically determined medium of Step 2, and cultured at 37°C for about 14 hours to prepare a bacterial solution.

[0037] Step 4) Inoculate 1% of strain 323-2 into 25 mL of the medium of step 2, and compare the effects of di...

Embodiment 3

[0040] Example 3: Optimization of sampling medium for fed-batch fermentation

[0041] Step 1) Prepare 5L of chemically determined medium, including 10g / L glucose, 2g / L(NH 4 ) 2 SO 4 , 4.2g / LKH 2 PO 4 And 11.24g / L K 2 HPO 4 , 1.7g / L citric acid, 0.5g / L MgSO 4 And 10mL / L trace element solution. Among them, the trace element solution contains 0.25g / L CoCl 2 ·6H 2 O, 1.5g / L MnSO 4 ·4H 2 O, 0.15g / L CuSO 4 ·2H 2 O, 0.3g / LH 3 BO 3 , 0.25g / L Na 2 MoO 4 ·2H 2 O,0.8g / L Zn(CH 3 COO) 2 , 5g / L iron (III) citrate and 0.84g / L EDTA, the pH of the trace element solution is 8.0.

[0042] Step 2) Sterilize the culture medium at 121°C for 15-20 minutes. (Note, the glucose solution should be sterilized separately and then mixed with other ingredients).

[0043] Step 3) Next, the strain 323-2 is pre-cultured in the medium with determined chemical composition in Step 2, and cultured at 37°C for about 14 hours to prepare a bacterial solution.

[0044] Step 4) Inoculate 2% strain 323-2 into the fermentor, us...

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Abstract

The invention relates to a construction method and a process of engineering bacteria for producing amorpha-4,11-diene. According to the strain for efficiently producing an artemisinin precursor amorpha-4,11-diene, recombinant bacteria are obtained by regulating the expression / or the activity of escherichia coli and an exogenous gene, wherein the gene comprises an acetoacetyl coenzyme A thiolase atoB gene, an HMG-CoA synthase hmgS gene, an HMG-CoA reductase hmgR gene, a mevalonate kinase mevK gene, a phosphohydroxyproline kinase pmk gene, a mevalonic acid-5-pyrophosphoric acid decarboxylase pmdgene, an isopentenyl pyrophosphate isomerase idi gene, a farnesyl pyrophosphate synthase ispA gene and an amorpha-4,11-diene synthase ads gene.

Description

Technical field [0001] The invention relates to a construction method and process of engineering bacteria for producing amorpha-4,11-diene (amorpha-4,11-diene). Background technique [0002] According to the World Health Organization, in 2010, 219 million people were infected with malaria and 660,000 died from malaria infections [World Health Organisation.World Malaria Report 2012 (WHO, 2012)]. Malaria comes from Plasmodium spp. parasites, mainly including Plasmodium falciparum and Plasmodium vivax. Among them, Plasmodium vivax has extensive resistance to traditional antimalarial drugs (such as chloroquine and sulfapolypyrimidine). Artemisinin and its derivatives are a new generation of fast-acting and low-toxic antimalarial drugs, and are one of the main components of the most effective antimalarial combination therapies (ACT). Artemisinin is an antimalarial drug extracted from the traditional Chinese medicine Artemisia annua by the Chinese pharmacist Tu Youyou in 1971. At the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P5/00C12R1/19
CPCC12N9/0006C12N9/1025C12N9/1085C12N9/1205C12N9/88C12N9/90C12P5/002C12Y101/01088C12Y203/0301C12Y205/0101C12Y205/01068C12Y207/01036C12Y401/01033C12Y402/03024
Inventor 张聪强
Owner 湖州爱蔻思生物科技有限公司
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