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Construction method and application of yeast recombinant strain for high-yield production of alpha-humulene

A technology for hopene and Candida tropicalis, which is applied in the field of metabolic engineering, can solve the problems of high extraction cost, complex plant metabolic pathways, and difficulty of α-hopene, and achieves simple product separation and purification steps and good industrial application. Prospects, the effect of avoiding dependence on raw materials

Active Publication Date: 2022-03-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the long growth cycle of plants, the content of α-humulene is greatly affected by the growth environment and climate, and the content of α-humulene in plants is low, so it is difficult to extract α-humulene from plant raw materials, and the extraction High cost
With the development of biological science and technology, the synthesis pathway of α-humulene in plants has been analyzed, but it is still difficult to produce α-humulene in large scale by plant tissue extraction.
On the one hand, due to the complex metabolic pathways of plants, genetic modification is difficult, on the other hand, environmental factors have a great impact on the content of α-humulene in plants, which is not conducive to industrial production

Method used

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  • Construction method and application of yeast recombinant strain for high-yield production of alpha-humulene
  • Construction method and application of yeast recombinant strain for high-yield production of alpha-humulene
  • Construction method and application of yeast recombinant strain for high-yield production of alpha-humulene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Construction of ZSS1 gene integrated expression cassettes and α-snakeae yeast strains H-01

[0074] 1. Construction of ZSS1 gene integrated expression box:

[0075] (1) According to the genomic sequencing result of the tropical faint yeast ATCC 20336 strain, the primers PPFA-F1 and PFBA-R1 (as shown in Table 2) are designed, and the chromosome DNA is chromosome DNA in tropical figs, and FBA1 is amplified by PCR. Gene promoter sequence (P FBA1 Nucleotide sequences are shown in SEQ ID NO.4; design primers TADH-F1 and TADH-R1 (as shown in Table 2), with tropical faint yeast ATCC 20336 chromosome DNA is a template, and an ADH2 gene is terminated by PCR. Subsequences;

[0076] (2) The ZSS1 gene (GenBank login number AB247331.1) is performed according to the ZsSs1 gene (GenBank login number AB247331.1), which is subjected to the primary chord of ZSS1 gene after chemical synthesis. (Nucleotide sequence, such as SEQ ID NO.1), the synthetic ZSS1 gene is inserted into the ...

Embodiment 2

[0084] Example 2: Construction of integrated expression cassettes in ZSS1 and ERG 20 and the construction of α-serpehloride yeast strains H-03

[0085] 1, ZSS1 and ERG20 simultaneously expressed the construction of integrated expression box:

[0086] (1) Design primers PGAP-F1 and PGAP-R1 according to the GAPDH gene sequence (GenBank login number HQ171163.1) in NCBI; the chromosome DNA is a template with a tropical faint yeast ATCC 20336, and GAPDH is amplified by PCR. Gene promoters (PGAP1 nucleotide sequences such as SEQ ID NO.5);

[0087] (2) According to the genomic sequencing result of the tropical faint yeast ATCC 20336 strain, the primer Erg20-F1 and ERG20-R1 are designed, and the chromosome DNA is chromosome DNA in the tropical fake silk yeast, and the Terrace Terminal Terrane T 7syn The fragment of the ERG20 gene (GenBank login number is RCK67668.1), then inserted into the PMD19-T Vector commercial carrier to obtain recombinant plasmid TM-ERG20-T 7syn ;

[0088] (3) PGAP1...

Embodiment 3

[0096] Example 3: Construction of alpha-serpentinic yeast strain H-07 and fermentation α-snake thimethylene

[0097] The primer sequence involved in this embodiment is shown in Table 3:

[0098] Table 3 primer sequence

[0099]

[0100]

[0101] Specific steps are as follows:

[0102] 1, ERG12, ERG8, ERG19 and IDi1 simultaneous expression of integrated expression box:

[0103] (1) According to the genomic sequencing result of the tropical faint yeast ATCC 20336 strain, the primer Erg12-F1 and ERG12-R1 are designed to be a template with a tropical faint yeast ATCC 20336 chromosome DNA, and the ERG12 gene sequence is amplified by the PCR (GenBank login number RCK63831.1), then inserted into the PMD19-T Simple commercial carrier to obtain recombinant plasmid TS-ERG12;

[0104] (2) Design primers PPFA-F1 and PFBA-R2, with tropical pseudo yeast ATCC 20336 chromosome DNA as a template, by PCR amplification of FBA1 gene promoters (P FBA1 The primer TPGK-F1 and TPGK-R1 were designed to...

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Abstract

The invention discloses a construction method and application of a yeast recombinant strain for high yield of alpha-humulene, and belongs to the field of metabolic engineering. According to the invention, an engineering strain capable of synthesizing alpha-humulene is constructed by integrating and expressing a plant-derived alpha-humulene synthase gene on chromosome DNA (Deoxyribonucleic Acid) of candida tropicalis, and an endogenous alpha-humulene synthesis gene is over-expressed on the basis, so that a recombinant strain capable of efficiently producing alpha-humulene is constructed; the yield of alpha-humulene reaches 149.7 mg.L <-1 > and is improved by 112 times compared with that of a control strain. The invention also provides a method for producing alpha-humulene by using the engineering strain. The alpha-humulene production strain provided by the invention is stable in production performance, does not need to add antibiotics in the production process, and has a good industrial application prospect.

Description

Technical field [0001] The present invention relates to the construction method of yeast recombinant strains of high yield alpha-serpene, which belongs to the field of metabolic engineering. Background technique [0002] Alpha-snake thiorene, also known as α-stone benyl or 葎葎ene is a monocyclic terpene existing in the grass plant. Studies have shown that snake thiis has an anti-inflammation, relieves pain, and effectively prevents viruses from respiratory diseases such as colds, pneumonia, and is very potential in anti-cancer. On the other hand, the hemea odor is particularly used, and it can also be used. In addition, snake thimethylene is also a basic skeleton of a plurality of terpene and terpenoids, which can produce a variety of high value-of-value products using snake thimethylene. [0003] Due to the long cycle of plant growth, the alpha-snake thimethylene content is larger than the growth environment and the climate, and the α-snake content in plants is low, so it is more...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/53C12N15/54C12N15/61C12N15/81C12N1/19C12P5/00C12R1/74
CPCC12N9/88C12N9/0006C12N9/1029C12N9/1025C12N9/1205C12N9/1229C12N9/90C12N9/1085C12N15/815C12P5/007C12Y402/03104C12Y101/01034C12Y203/01009C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y205/0101C12N2800/102Y02A50/30
Inventor 张利华陈献忠沈微夏媛媛杨海泉
Owner JIANGNAN UNIV
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