Recombinant escherichia coli for producing coniferyl alcohol by taking eugenol as substrate and application of recombinant escherichia coli
By constructing recombinant Escherichia coli and using eugenol as a substrate, the efficient synthesis of coniferyl alcohol was successfully achieved, solving the problems of low efficiency and high cost of coniferyl alcohol production in traditional methods, and achieving high conversion rate and large-scale production.
Patent Information
- Application Number
- CN202510800125.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-09-16
AI Technical Summary
In the existing technology, the acquisition of coniferyl alcohol mainly relies on extraction from natural plants, which has the problems of high cost, low efficiency and being restricted by the plant growth cycle and geographical environment, making it difficult to achieve large-scale and stable supply.
By constructing a recombinant Escherichia coli, introducing vanillyl alcohol oxidase and peroxidase genes, and using eugenol as a substrate, the synthesis of coniferyl alcohol was catalyzed during the fermentation process, and the yield was increased by optimizing the fermentation conditions.
The efficient synthesis of coniferyl alcohol was achieved, with a yield of 23.17±0.41 g/L and a eugenol conversion rate of up to 92.68%, breaking through the limitations of traditional methods and providing an efficient and sustainable production route.
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Figure CN120648714A_ABST
Abstract
Claims
1. A method for constructing a recombinant Escherichia coli for producing coniferyl alcohol using eugenol as a substrate, characterized in that: The following steps are involved: (1) The Amvao gene with the nucleotide sequence shown in SEQ ID NO. 18 and the pspAPX1 gene with the nucleotide sequence shown in SEQ ID NO. 21 were ligated into the PET-30a plasmid to obtain the pET30a-AmvaoA-PspAPX1 plasmid; (2) The pET30a-AmvaoA-PspAPX1 plasmid in step (1) is transformed into Escherichia coli to obtain a recombinant Escherichia coli that can produce high levels of coniferyl alcohol using eugenol as a substrate.
2. The construction method according to claim 1, characterized in that The specific operations of step (1) are as follows: ① Linearize the vector PET30a by inverse PCR using primers with sequences as shown in SEQ ID NOs. 1 and 2; ② Amplify the Amvao gene using primers with sequences as shown in SEQ ID NO. 5 and 6 to obtain a linearized fragment; ③ The two linearized fragments in steps ① and ② were cloned and infused to obtain the recombinant plasmid pET30a-AmvaoA; ④ Amplify the pspAPX1 gene using sequences such as SEQ ID NO. 11 and 12 to obtain a linearized fragment; ⑤ Linearize the recombinant plasmid pET30a-AmvaoA by inverse PCR using primers with sequences as shown in SEQ ID NOs. 9 and 10; ⑥The two linearized fragments in steps ④ and ⑤ were cloned and infused seamlessly to obtain the pET30a-AmvaoA-PspAPX1 plasmid.
3. The construction method according to claim 2, characterized in that The Escherichia coli is BL21 (DE3).
4. The recombinant Escherichia coli obtained by the construction method according to any one of claims 1 to 3.
5. A method for producing coniferyl alcohol by fermentation using the recombinant Escherichia coli according to claim 4, characterized in that: The following steps are involved: (1) preparing a seed solution of recombinant Escherichia coli; (2) inoculating the seed solution in step (1) into M9YE medium and culturing at 37°C with shaking for 14-18 hours; (3) Add 20-30 g / L eugenol as a substrate and continue fermentation for 90-100 h.
6. The method according to claim 5, characterized in that The shaking culture time in step (2) is 16 hours.
7. The method according to claim 6, characterized in that The concentration of eugenol in step (3) is 25 g / L.
8. The method according to claim 7, characterized in that The fermentation time in step (3) is 96 hours.
9. Use of the recombinant Escherichia coli according to claim 4 in the fermentation preparation of coniferyl alcohol.
10. Use of the method according to any one of claims 5 to 8 in the fermentation preparation of coniferyl alcohol.