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A kind of Saccharomyces cerevisiae engineering bacteria producing dihydroartemisinic acid and its construction method and application

A technology of dihydroartemisinic acid and Saccharomyces cerevisiae, applied in the field of microorganisms, can solve the problems of affecting the production of target substances and the high proportion of artemisinic acid

Active Publication Date: 2021-08-24
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the technology of directly synthesizing dihydroartemisinic acid by using microorganisms heterogeneously will not produce chiral dihydroartemisinic acid as a by-product, but it will generate artemisinic acid as a by-product at the same time, as described in the existing patent CN201610876830.X The ratio of dihydroartemisinic acid / artemisinic acid in the constructed recombinant strain is only 2.53, which indicates that the proportion of artemisinic acid is too high, which affects the production of target substances

Method used

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  • A kind of Saccharomyces cerevisiae engineering bacteria producing dihydroartemisinic acid and its construction method and application
  • A kind of Saccharomyces cerevisiae engineering bacteria producing dihydroartemisinic acid and its construction method and application
  • A kind of Saccharomyces cerevisiae engineering bacteria producing dihydroartemisinic acid and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Construction and import of module hphA

[0067] Using the plasmid SyBE_Ec01130018 as a template, fragment H0 (hphMX coding sequence) was amplified with 17E0b-hphA-F and 17E0b-hphA-R as primers; Hup was added (the homology arm 1 near the upstream His3 of the original ALDH1 coding sequence was used as the upstream homology arm); using the genome of SyBE_Sc01130057 as a template, Hdown was amplified with primers 17E0down-F and 17E0down-R (the original ALDH1 T downstream of the coding sequence TDH1 , as the downstream homology arm); then use the OE-PCR method to amplify the three fragments of H0, Hup, and Hdown using 17E0up-F, 17E0down-R as primers to amplify the module hphA, that is, the homology arm 1 (upstream of the His3 tag Partial sequence)+hphMX+T TDH1 . hphA was introduced into the strain SyBE_Sc01130057, and the strain SyBE_Sc01130352 was obtained through homologous recombination in yeast; the construction diagram will be figure 2 , see the import d...

Embodiment 2

[0068] Example 2: His3 tag (containing homology arm 1, that is, using the partial sequence upstream of the His3 tag as homology arm 1)+P GAL7 +ALDH1 H194R / ALDH1 V247F The coding sequence of +T TDH1 The construction and import of the module

[0069] 1. His3 tag +P GAL7 +ALDH1 V247F The coding sequence of +T TDH1 The construction and import of the module

[0070] The plasmid pSB1C3 was treated with EcoRI and PstI to obtain the fragment Vector-EP; using the genome of the strain SyBE_Sc01130057 as a template, the fragment E-His3, namely the His3 tag, was amplified by PCR with primers 17E1-His3-FE and 18E-His3-R;

[0071] Using SyBE_Ec01130018 as a template, the fragment E-ALDcF-a, namely P GAL7 +ALDH1 V247F The upper half of the coding sequence; using primers 18E5cF-F and 17E1-ALDH1-RP, the fragment E-ALDcF-b, namely ALDH1, was amplified by PCR V247F The lower half of the coding sequence;

[0072] The fragment E-ALDcF-a and fragment E-ALDcF-b were connected by OE-PCR an...

Embodiment 3

[0083] Example 3: His3 tag (containing homology arm 1, that is, using the partial sequence upstream of the His3 tag as homology arm 1)+P GAL7 +DBR2-ALDH1 H194R The coding sequence of +T TDH1 The construction and import of the module

[0084] The plasmid pSB1C3 was treated with EcoRI and PstI to obtain the fragment Vector-EP; using the plasmid pRS423 as a template, the fragment E-His3, namely the His3 tag, was amplified by PCR with primers 17E1-His3-FE and 17E1-His3-R;

[0085] Using SyBE_Ec01130021 as a template, the fragment E-DBR2, namely P GAL7 + DBR2 coding sequence;

[0086] Using SyBE_Ec01130018 as a template, using primers 17E1z-ALDH1-F and 18E5dR-R, the fragment E-ALD-1, ALDH1 was amplified by PCR H194R The lower half of the coding sequence; with SyBE_Ec01130018 as a template, using primers 18E5dR-F and 17E1-ALDH1-RP, the fragment E-ALD-2, ALDH1 was amplified by PCR H194R The upper half of the coding sequence;

[0087] Fragment E-His3 and fragment E-DBR2 were con...

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Abstract

The invention relates to the technical field of microbes, and discloses a Saccharomyces cerevisiae engineering bacterium for producing dihydroartemisinic acid. The engineering bacteria of the present invention are based on the Saccharomyces cerevisiae modified by the heterologous synthesis pathway of S. cerevisiae dihydroartemisinic acid, so that the original sequence expressing ALDH1 is ALDH1 expressing mutation H194R or ALDH1 V247F the sequence of. The present invention performs site-directed mutation on the key gene ALDH1 of the pathway to obtain a gene with better selectivity, so that the metabolic pathway prefers the synthesis of dihydroartemisinic acid and reduces the proportion of artemisinic acid. Two mutants of ALDH1 H194R and ALDH1 V247F It was demonstrated in the present invention that the dihydroartemisinic acid / artemisinic acid ratio can be increased. On this basis, the fusion expression of DBR2 and ADH1, and the fusion expression of mutant ALDH1 and DBR2 can further increase the ratio of dihydroartemisinic acid / artemisinic acid without sacrificing the production of dihydroartemisinic acid.

Description

technical field [0001] The invention relates to the technical field of microorganisms, and more specifically relates to a Saccharomyces cerevisiae engineered bacterium producing dihydroartemisinic acid and its construction method and application. Background technique [0002] Artemisinin is an effective antimalarial drug, and many of its derivatives were identified as first-line antimalarial drugs by the World Health Organization in 2002. At present, artemisinin is mainly obtained directly from the plant Artemisia annua. Artemisia annua is distributed all over the world, but the content of artemisinin in most of them is very low (≤1‰), and the extraction cost is high. The de novo synthesis of artemisinin by chemical synthesis is economically unfeasible due to the complex structure of artemisinin molecules, which makes the synthesis difficult and costly. The biosynthesis method is the best choice to obtain artemisinin, that is, through genetic engineering to transform micro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P7/40C12R1/865
CPCC12P7/40C12N9/0008C12Y102/0101C12N15/81Y02A50/30
Inventor 元英进曾薄轩肖文海姚明东王颖
Owner TIANJIN UNIV
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