Construction method of gene engineered bacteria for arteannuic acid production

A technology of genetically engineered bacteria and artemisinic acid, applied in the field of microbial fermentation, can solve the problems of relying on the selection of enzyme cutting sites, high cost, and great difficulty, and achieve good industrial application prospects, high efficiency of homologous recombination, and shorten construction time Effect

Inactive Publication Date: 2014-08-13
SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The second method is to produce artemisinin through chemical total synthesis. This method is costly and difficult. It cannot be put into production at present, and it is basically in the experimental stage.
[0013] According to the above literature, although artemisinic acid genetically engineered bacteria can be constructed, the genes related to artemisinic acid expression need to be integrated into the yeast chromosome in sequence, which involves multi-stage cloning and depends on the selection of suitable restriction sites , the operation is cumbersome and the cycle is long

Method used

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  • Construction method of gene engineered bacteria for arteannuic acid production
  • Construction method of gene engineered bacteria for arteannuic acid production
  • Construction method of gene engineered bacteria for arteannuic acid production

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Experimental program
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Embodiment 1

[0037] The experimental scheme design of this embodiment is as follows:

[0038] In Saccharomyces cerevisiae BY4742 (purchased from EUROSCARF, No. Y10000, strain information: MATα; his3△1; leu2△0; lys2△0; ura3△0) expressing three genes ads from the artemisinin synthesis pathway of Artemisia annua (GenBank: DQ241826), amo (GenBank: DQ872632) and cpr (GenBank: DQ984181), optimized by yeast preference codons, and simultaneously expressing the ble gene (Zeocin resistance gene) as a selection marker, and selecting the yeast chromosome δ site for integration site, the integration and assembly sequence of each gene module on the chromosome is as follows: figure 1 shown. The promoters and genes are numbered respectively, as shown in Table 1.

[0039] Table 1. Promoters and gene numbers

[0040] promoter number promoter name gene number gene name A TEF1p 1 ads

[0041] B TPI1p 2 amo C PGK1p 3 cpr

[0042]The following gene ex...

Embodiment 2

[0051] 2.1. Construction of plasmids pLYADS, pLYAMO and pLYCPR

[0052] References (Yang RY, Feng LL, Yang XQ, Yin LL, Xu XL, Zeng QP. Quantitative transcript profiling reveals down-regulation of A sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants. Planta medica, 2008Oct ;74(12):1510-6.Epub2008Sep24.) Ads, amo and cpr genes were obtained, optimized and synthesized by yeast preferred codons (SEQ ID No: 29-31), and inserted into pMD19-T Simple Vector (purchased from Takara company), the plasmids pLYADS, pLYAMO and pLYCPR were obtained and transformed into E.coli strain DH5α for preservation.

[0053] 2.2. Extraction of template DNA

[0054] The extraction method of Saccharomyces cerevisiae BY4742 chromosomal DNA is as follows: pick a plump single colony and connect it to a 3ml YPD liquid medium test tube (20ml shake flask), 30°C, 220rpm / min, cultivate overnight for about 18h, collect the bacterial liquid, and us...

Embodiment 3

[0078] 3.1. Fermentation

[0079] The seed cultivation method is as follows:

[0080] Pick a single colony of the positive clone obtained from the screening in Example 2 from the culture plate, insert it into 25ml of YPD medium (250ml shake flask), culture at 30°C, 220rpm / min, for 48h.

[0081] The fermentation culture method is as follows:

[0082] The seed liquid was added to 25ml YPD medium (250ml shake flask) according to the inoculation ratio of 4%, and cultured at 30°C and 220rpm / min for 120h.

[0083] 3.2. Extraction of artemisinic acid

[0084] The extraction method of artemisinic acid is as follows:

[0085] Take an appropriate amount of fermentation broth, adjust the pH to 8.5-9.0, dilute with methanol to the required multiple (such as 50 times), transfer the mixed solution into a centrifuge tube, centrifuge at 4000r / min for 30min, and collect the supernatant.

[0086] 3.3. Detection of artemisinic acid

[0087] Refer to the literature method (Zhang Dong et al.,...

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Abstract

The invention discloses a construction method of gene engineered bacteria for arteannuic acid production, and the construction method comprises the following steps: A) construction of a gene expression module including an artemisinic acid production related gene, an upstream promoter of the artemisinic acid production related gene, and a downstream terminator of the artemisinic acid production related gene; and B) co-transformation of the gene expression module into saccharomyces cerevisiae. The method has the advantages of being simple and fast and efficient, avoids multiple cloning, and does not rely on enzyme digestion sites, many fragments can be together transformed, the homologous recombination efficiency is high, and the method can significantly shorten the construction time of the gene engineered bacteria.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a construction method of a genetically engineered bacterium producing artemisinic acid. Background technique [0002] Artemisinin is an effective antimalarial monomer isolated and purified from the traditional Chinese herbal medicine Artemisia annua (Artemisia annua L.) by Chinese scientists in the 1970s. Its chemical essence contains a "peroxide bridge" structure ( 1,2,4-trioxane ring) sesquiterpene lactone. Artemisinin succinate (artesunate), artemisinin ether (artemether), artemisinin ether (artemisinin) and dihydroartemisinin obtained by artificial semi-synthesis with artemisinin as the core Artemisinin and other artemisinin drugs have good solubility in blood and high bioavailability, and have special effects on chloroquine-resistant malaria and fatal cerebral malaria. Artemisinin-based combination therapies (ACTs) are the preferred antimalarial new drugs,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12R1/865
Inventor 魏维戈梅孙新强罗敏玉肖彩霞夏兴盛保伟金一平
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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