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Saccharomyces cerevisiae engineering bacteria for producing dihydroarteannuic acid and construction method and application thereof

A technology of dihydroartemisinic acid and Saccharomyces cerevisiae, which is applied in the field of microorganisms, can solve the problems of high proportion of artemisinic acid, influence the generation of target substances, etc., and achieve the effect of increasing the proportion

Active Publication Date: 2020-04-28
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. 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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  • Saccharomyces cerevisiae engineering bacteria for producing dihydroarteannuic acid and construction method and application thereof
  • Saccharomyces cerevisiae engineering bacteria for producing dihydroarteannuic acid and construction method and application thereof
  • Saccharomyces cerevisiae engineering bacteria for producing dihydroarteannuic acid and construction method and application thereof

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 bacterial 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...

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] Treat the plasmid pSB1C3 with EcoRI and PstI to obtain the fragment Vector-EP; use the plasmid pRS423 as a template, and use primers 17E1-His3-FE and 17E1-His3-R to amplify the fragment E-His3, which is the His3 tag, by PCR;

[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 connected...

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Abstract

The invention, which relates to the technical field of microorganisms, discloses saccharomyces cerevisiae engineering bacteria for producing dihydroarteannuic acid. On the basis of saccharomyces cerevisiae modified by a dihydroarteannuic acid saccharomyces cerevisiae heterologous synthesis route, the original ALDH1 expression sequence is an ALDH1H194R or ALDH1V247F sequence with expression mutation. According to the invention, site-specific mutagenesis is carried out on a key gene ALDH1 of a path to obtain a gene with better selectivity, so that a metabolic path better prefers synthesis of dihydroartemisinic acid and the proportion of artemisinic acid is reduced. The two mutants ALDH1H194R and ALDH1V247F are proved to be capable of increasing the ratio of dihydroarteannuic acid to arteannuic acid in the invention. Therefore, DBR2 and ADH1 are subjected to fusion expression; meanwhile, mutants ALDH1 and DBR2 are subjected to fusion expression, so that the ratio of dihydroartemisinic acid to artemisinic acid is further increased, and the yield of dihydroartemisinic acid is not sacrificed.

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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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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