Method for reforming terpene-based synthase

A technology of terpene synthase and nucleic acid molecules, which is applied in the biological field and can solve problems such as the development of terpene synthase

Active Publication Date: 2018-07-03
WUHAN HESHENG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, terpene synthases are still to be developed

Method used

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  • Method for reforming terpene-based synthase
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  • Method for reforming terpene-based synthase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] A multiple sequence alignment was performed on the sequences of 51 fungal terpene synthases whose functions were clearly verified in the literature reports before August 2016. Subsequently, the substrate-binding regions of these proteins were analyzed in combination with the representative crystal structure data, and the highly conserved catalytic-related sites were excluded to obtain the amino acid sites that determine functional specificity (Table 1). These correspond to the 65th, 69th, 85th, 88th, 89th, 159th, 186th, 191st, 219th, 222nd, 307th, 311th, 314th positions in the terpene synthase FgMS, There are 13 positions in total. The gray part is the aromatic amino acid.

[0070] Table 1 Amino acid positions of terpene synthase

[0071]

[0072]

Embodiment 2

[0073] Example 2 Transformation of phenylalanine at position 65 of wild-type terpene synthase FgMS into leucine

[0074] In this example, the wild-type FgMs terpene synthase was mutated according to the following method, so that compared with the wild-type terpene synthase, the 65th phenylalanine was transformed into leucine :

[0075] The mutant gene F65L was obtained by mutating the 193rd-195th nucleotides of the nucleotide sequence shown in SEQ ID NO: 6 from TTT to CTG. After the gene was obtained, it was cloned into the expression vector of pet28a, and transformed into the expression host E.coli BL21(DE3). After transformation, single clones were picked and placed in LB medium containing corresponding antibiotics, and cultured overnight at 37°C and 220rpm. Transfer to 1L of fresh LB medium containing corresponding antibiotics according to 1% inoculum amount, and cultivate to OD at 37°C and 220rpm 600 About 0.6-0.8, lower the temperature to 16°C, add IPTG with a final co...

Embodiment 3

[0080] Example 3 Transformation of phenylalanine at position 89 of wild-type terpene synthase FgMS into leucine

[0081] According to the method of Example 2, the 265th-267th nucleotide of the nucleotide sequence shown in SEQ ID NO: 6 is mutated from TTT to CTG, so that the obtained terpene synthase is compared with the wild-type terpene synthase, The phenylalanine at position 89 was transformed into leucine.

[0082] Verification is carried out according to the mode of embodiment 2, and the results are shown in figure 1 (2), the following changes occurred in the product: 1. The content ratio of sesquiterpene main product 1 increased from 92.82% to 100%; 2. The content ratio of the original diterpene by-product 35 increased from 1.90% to 49.50%, becoming the main product.

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PUM

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Abstract

The present invention provides terpene-based synthase, a nucleic acid molecule, a construction body, recombinant cells and uses of the terpene-based synthase, the nucleic acid molecule, the construction body and the recombinant cells, and a terpene-based synthase obtaining method. Compared to the wild type terpene-based synthase, the terpene-based synthase of the present invention has at least oneof the following mutations that the aromatic amino acid in the hydrophobic or neutral amino acid in the active region is mutated into the non-aromatic amino acid, and the non-aromatic amino acid in the hydrophobic or neutral amino acid in the active region is mutated into the aromatic amino acid. According to the present invention, under the catalytic effect of the terpene-based synthase of the present invention, the abundance of the main product in the catalytic product can be changed, and the original by-product can be changed into the main product or the new terpene-based compound can be obtained.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to a method for modifying terpene synthase. Background technique [0002] Terpenoids are a general term for compounds containing isoprene units. So far, people have discovered about 76,000 kinds of terpenoids from animals, plants and microorganisms. It is widely used in perfume production industry, health care product industry, agricultural production field and medical industry. [0003] However, terpene synthases are currently still to be developed. Contents of the invention [0004] The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. Therefore, the present invention proposes a terpene synthase, a nucleic acid molecule, a construct, a recombinant cell, use and a method for obtaining the terpene synthase. [0005] It should be noted that the present invention is based on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60
CPCC12N9/88
Inventor 刘天罡卞光凯韩以超侯安伟苑玉杰刘然
Owner WUHAN HESHENG TECH CO LTD
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