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Tropinone biosynthesis fusion protein and its application and method

A fusion protein and biosynthesis technology, applied in the field of metabolic engineering, can solve problems such as limiting the accumulation of downstream medicinal tropane alkaloids, and achieve the effects of improving synthesis efficiency and improving synthesis ability

Active Publication Date: 2022-04-29
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previous studies have shown that tropinone biosynthesis is the key rate-limiting step in the biosynthesis of tropane alkaloids in both plant metabolic engineering and yeast synthetic biology, which greatly limits the downstream medicinal tropane alkaloids. accumulation

Method used

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  • Tropinone biosynthesis fusion protein and its application and method
  • Tropinone biosynthesis fusion protein and its application and method
  • Tropinone biosynthesis fusion protein and its application and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, discovery of tropinone biosynthesis metabolic compartment

[0030] (1) Extraction of total RNA from belladonna fibrous root

[0031] Take an appropriate amount of belladonna fibrous root tissue, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and the RNA concentration was determined on a spectrophotometer.

[0032] (2) Gene cloning

[0033] Using the extracted total RNA as a template, synthesize cDNA according to the instructions of the Tiangen FastKing cDNA First Strand Synthesis Kit; design gene-specific primers, and the specific primers are as follows:

[0034] F-CYP82M3: 5'-cgcatgtatgataattttctcttctatga-3' (SEQ ID NO.1);

[0035] R-CYP82M3: 5'-cgcaaattcataaagcacagaattc-3' (SEQ ID NO.2);

[0036] F-PYKS: 5'-cgcatgaagttggaaaatgg...

Embodiment 2

[0046] Embodiment 2, the construction of tropinone biosynthesis fusion protein

[0047] In order to construct the biosynthetic fusion protein of tropinone, the primer sequences were designed as follows:

[0048] F-3xGGGGS-CYP82M3:

[0049] 5'-ggtggggggaggtccgggggtggggggagtgggggaggtgggtcaatgtatgataatttt-3' (SEQ ID NO. 7);

[0050] R-CYP82M3-3xGGGGS:

[0051] 5'-tgacccacctcccccactccctccaccccccggaccctcccccaccaaattcataaagca-3' (SEQ ID NO. 8);

[0052] F-3xGGGGS-PYKS:

[0053] 5'-ggtggggggaggtccgggggtggggggagtgggggaggtgggtcaatgaagttggaaa-3' (SEQ ID NO. 9);

[0054] R-PYKS-3xGGGGS:

[0055] 5'-tgacccacctcccccactccctccaccccccggaccctccccccaccaatgggcacactacg-3' (SEQ ID NO. 10);

[0056] The CYP82M3-3xGGGGS-PYKS fusion gene was obtained by overlapping PCR, and the specific steps were as follows: the PYKS gene cloned in the previous step was used as a template, and primers F-3xGGGGS-PYKS (SEQ ID NO.9) and R-PYKS (SEQ ID NO. 4), perform PCR amplification, and recover by cutting the...

Embodiment 3

[0058] Example 3, Evaluation of Tropinone Biosynthetic Fusion Protein in Plant Metabolic Engineering

[0059] (1) Construction of plant overexpression vector

[0060] In order to evaluate, PYKS single gene expression, CYP82M3 single gene expression, CYP82M3 / PYKS double gene co-expression, CYP82M3-3xGGGGS-PYKS fusion gene expression, PYKS-3xGGGGS-CYP82M3 fusion gene expression, five combinations in the plant medicinal tropane biological In view of the application value in alkali metabolism engineering, the above five plant expression plasmids were constructed in this study. Using the above-mentioned genes as templates, KOD-Plus was used to amplify the genes, and restriction endonucleases and T4 DNA ligase were used to carry out enzyme-linking reactions.

[0061] With the plant binary expression vector pBI121 and pCAMBIA1305.1 as the original plasmid, the selection marker gene of pBI121 is the kanamycin (NPTII) gene, and the selection marker gene of pCAMBIA1305.1 is the hygromy...

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Abstract

The invention discloses a tropinone biosynthesis fusion protein and its application and method. The tropinone biosynthesis fusion protein is connected by a type III polyketide synthase PYKS and a tropinone synthase CYP82M3 through a connecting peptide. The type III polyketide The synthase PYKS is located at the carboxyl terminal of the connecting peptide, and the tropinone synthase CYP82M3 is located at the amino terminal of the connecting peptide; in the present invention, PYKS and CYP82M3 are fused and expressed to interact to form the metabolic compartment of tropinone. The synthesis efficiency of tropinone is improved, and its effect is better than that of PYKS and CYP82M3 for double-gene co-expression, or the fusion protein of CYP82M3 located at the carboxyl end of the connecting peptide, so this fusion protein can be used to improve the synthesis ability of tropinane alkaloids, which is beneficial to tropinone The production of pinane alkaloids is of great significance.

Description

technical field [0001] The invention relates to the field of metabolic engineering, in particular to a fusion protein for biosynthesis of tropinone, and also relates to the application of the fusion protein for biosynthesis of tropinone and a method for increasing tropine alkaloids. Background technique [0002] Tropane alkaloids (Tropane Alkaloid, TA) are a class of natural anticholinergic drugs with great medical value, widely used in anesthesia, analgesia, cough, asthma and anti-motion sickness, and also used to control stiffness in Parkinson's disease and tremors. Hyoscyamine and scopolamine are commonly used clinically, and the market demand is huge. At present, TA is extracted from a few TA resource plants of the family Solanaceae, including Atropa belladonna, Daturastramonium and Hyoscyamus niger. It is also a TA drug source plant included in the Pharmacopoeia. The mass fraction of scopolamine in wild belladonna plants is 0.02%-0.17% (dry weight), and the content o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12P17/18
CPCC12N9/1029C12N9/00C12P17/182C07K2319/00
Inventor 曾俊岚廖志华邱飞刘雪超陈敏杨春贤
Owner SOUTHWEST UNIV
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