Recombinant strain containing enol kinase gene and isopentenyl phosphokinase gene and application of recombinant strain in terpenoid production

A technology for isopentenyl phosphokinase and terpenoids, which is applied in the application field of terpenoid production, can solve problems such as enhancing the metabolic flux of precursors, and achieves the effects of facilitating rapid accumulation, efficient utilization, and increased yield

Pending Publication Date: 2022-04-29
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The natural synthesis pathways of IPP and DMAPP are the mevalonate pathway (MVA) and the methylerythritol 4-phosphate pathway (MEP), which are closely related to and dynamically regulated by the central carbon metabolism of the cell. Constrained by carbon sources, low energy efficiency, and other metabolic pathways in organisms, it is impossible to fundamentally solve the problem by using natural synthetic pathways as an entry point to enhance the metabolic flux of precursors

Method used

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  • Recombinant strain containing enol kinase gene and isopentenyl phosphokinase gene and application of recombinant strain in terpenoid production
  • Recombinant strain containing enol kinase gene and isopentenyl phosphokinase gene and application of recombinant strain in terpenoid production
  • Recombinant strain containing enol kinase gene and isopentenyl phosphokinase gene and application of recombinant strain in terpenoid production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Construction of yeast chassis for heterologous synthesis of tetraterpenoids (lycopene)

[0058] with high-fidelity enzymes ( Max Super-Fidelity DNA Polymerase) PCR to obtain the promoter (P GAL1 ,P GAL10 ,P GAL7 ), terminator (T ADH1 , T CYC1 , T TDH2 ) and the heterologous genes CrtE (geranylgeranyl pyrophosphate synthase, GGPP synthase), CrtB (phytoene synthase) and CrtI (phytoene dehydrogenase). By fusion PCR, the above elements were assembled into P GAL10 -CrtE-T ADH1 ,P GAL1 -CrtB-T CYC1 ,P GAL7 -CrtI-T TDH2 And construct the exogenous gene expression box CrtE-CrtB-CrtI, the structure is as follows figure 2 shown. Gel cutting and recovery to obtain the purified target fragment.

[0059] Using the CRISPR / Cas9 tool, the above exogenous gene expression cassette CrtE-CrtB-CrtI was integrated into the GAL80 position of the wild-type Saccharomyces cerevisiae CEN.PK2-1C genome to obtain the terpene-producing yeast chassis.

[0060] The knock-in...

Embodiment 2

[0073] Example 2: Assembly and expression of IAP pathway functional elements

[0074] Enol kinase (IAK) gene, phosphokinase (IPK) gene and bidirectional strong promoter P GAL10-GAL1 Assembled into an IAK-IPK expression cassette. The IAK-IPK expression cassette was recombined with the low-copy vector pRS415-LEU2 (CEN / ARS) using Gibson assembly technology to obtain the recombinant plasmid pRS415-IAK-IPK (structure as Figure 4 shown). These recombinant plasmids were transformed into the above-mentioned terpene-producing yeast chassis GLy80 for expression, and the transformation method was the same as in the above-mentioned Example 1.

[0075] The recombinant plasmids constructed in this example are: pRS415-ScCK-AtIPK and pRS415-SeCK-CsIPK.

[0076] Wherein, the specific genotype in the pRS415-ScCK-AtIPK plasmid combination is: the ScCK gene derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) (the nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid se...

Embodiment 3

[0079] Example 3: Application of the Isopentenol Pathway (IAP) in Yeast Chassis

[0080] The yeast transformed in the above-mentioned Example 2 was spread on the plate for about 3 days to grow pink single colonies.

[0081] (1) Pick a single clone in a sterilized 24-well plate, load 2 mL of amino acid-deficient medium (SC-LEU+2% glucose) in each well of the 24-well plate, and culture overnight at 30° C., 800 rpm.

[0082] (2) Transfer the next day, take a certain amount of the above bacterial solution into a new 24-well plate. As above, each well of a 24-well plate was loaded with 2 mL of amino acid-deficient medium (SC-LEU+2% glucose). The initial OD600 after transfer was 0.2; cultured at 30°C, 800rpm for 24 hours.

[0083] (3) After 24 hours, it is measured that the above-mentioned bacterium grows to about OD600 of about 4-5, and the enol substrate Isoprenol and (or) Prenol are added to the medium with a final concentration of 25 mM, and then 10% dodecane ( V / V), that is,...

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Abstract

The invention discloses a recombinant strain containing an enol kinase gene and an isopentenyl phosphokinase gene and application of the recombinant strain in terpenoid production. The recombinant strain comprises an enol kinase gene and an isopentenyl phosphokinase gene, and preferably, the recombinant strain is selected from saccharomycetes or escherichia coli. The invention also discloses a recombinant plasmid containing an enol kinase gene and an isopentenyl phosphokinase gene, a construction method of the recombinant plasmid, and a method for producing terpenoids by using the recombinant strain. The gene is constructed on a shuttle plasmid or integrated on a genome to express enol kinase and isopentenyl phosphokinase in recombinant bacteria, and isopentenyl pyrophosphoric acid and dimethyl allyl pyrophosphoric acid are produced by taking enol as a substrate to participate in synthesis of terpenoids. According to the approach, only two-step reaction and participation of a cofactor (ATP) are needed, and the approach is separated from central carbon metabolism; according to the invention, the biosynthesis process of the terpenoids has stronger metabolic flux, and the yield of the terpenoids is improved.

Description

technical field [0001] The invention relates to the field of microbial genetic engineering, in particular to a recombinant bacterial strain containing an enol kinase gene and a prenyl phosphokinase gene and its application in the production of terpenoids. Background technique [0002] Terpenoids are a class of compounds with a wide variety of functions and are widely used in various fields such as medicine, agriculture, biofuels, spices and health products. They have important physiological activities and high economic and medical strategic value. Some terpenoids are already approved clinical drugs, and many are in clinical testing. Researchers have made great progress in using microorganisms to produce terpenes, which is currently recognized as the most effective way of low-cost, high-efficiency, and sustainable production. Although the heterologous synthesis of various terpenes has been realized in engineering bacteria, the yield is low and cannot meet the huge market dem...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/54C12N9/12C12N1/19C12P5/02C12R1/865
CPCC12N15/81C12N9/1229C12N9/12C12Y207/04026C12P5/007
Inventor 罗小舟周小雪沈俊峰
Owner SHENZHEN INST OF ADVANCED TECH
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