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Olivol synthase variants and engineered microorganisms expressing them

An olivetolic acid cyclase and engineering technology, applied in the field of microorganisms and enzymes, can solve the problems of difficult to obtain high-purity products, harsh conditions, limited production capacity, etc.

Active Publication Date: 2022-01-07
BLUEPHA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the natural extraction of olivetol and olivelic acid from plants
Moreover, plant planting is limited by many factors, for example, it is more difficult to obtain high-purity products than biosynthesis, strict planting controls, limited production capacity, high investment in plant planting and downstream extraction, and low production stability
The chemical synthesis of olive alcohol and olive oil is expensive, and there are problems such as environmental pollution and harsh conditions
For the biosynthesis of olivetol and oliveolic acid, according to the knowledge of the inventors, the yield of biosynthetic olivetol and olivetolic acid is not enough to meet the requirements of industrialization with Escherichia coli as the chassis bacteria

Method used

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  • Olivol synthase variants and engineered microorganisms expressing them
  • Olivol synthase variants and engineered microorganisms expressing them
  • Olivol synthase variants and engineered microorganisms expressing them

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0067] Item 1. An engineered Escherichia coli, wherein the engineered Escherichia coli is modified to express an olivetol synthase (OLS) variant, wherein the OLS variant comprises one of the following mutations compared to its wild type One or more: I303T, I52L, S56A, H262M and K263R.

[0068] Item 2. The engineered E. coli according to any one of the preceding items, wherein the engineered E. coli is modified to express olivate cyclase (OAC).

[0069] Item 3. The engineered Escherichia coli according to any one of the preceding items, wherein the genomic fabH gene of the engineered Escherichia coli is deleted.

[0070] Item 4. The engineered Escherichia coli according to any one of the preceding items, wherein the genome fadE gene of the engineered Escherichia coli is deleted.

[0071] Item 5. The engineered E. coli according to any one of the preceding items, wherein both the fabH and fadE genes of the engineered E. coli genome are deleted.

[0072] Item 6. The engineered ...

Embodiment 1

[0101] Example 1: Deletion of the fabH gene

[0102] The fabH gene in the genome of Escherichia coli BW25113 was deleted to reduce the flow of intracellular Malonyl-CoA to the branch metabolism, thereby increasing the accumulation of intracellular Malonyl-CoA to further increase the synthesis of the target product OA.

[0103] Design and synthesize the H1-kana-H2 described in SEQ ID NO: 1, according to the literature (Datsenko K A, Wanner BL. One-step inactivation of chromosome genes in Escherichia coli K-12 usingPCR products[J]. Proceedings of the National Academy of Sciences, 2000, 97 (12): 6640-6645.) provided a method to integrate SEQ ID NO: 1 into the fabH gene position of the genome of BW25113 through λ-Red homologous recombination to delete the fabH gene, the steps are as follows:

[0104] 1. Prepare the competent state of BW25113;

[0105] 2. Introduce plasmid pKD46;

[0106] 3. Inoculate BW25113 (pKD46) into 3 mL of LB containing ampicillin, the concentration of a...

Embodiment 2

[0126] Example 2: Deletion of the fadE gene

[0127] In the engineered BW25113 obtained in Example 1, the fadE gene in the genome was further deleted to reduce the flow of intracellular hexanoyl-CoA to the bypass metabolism, thereby increasing the accumulation of intracellular hexanoyl-CoA to further increase the synthesis of the target product OA .

[0128] Design and synthesize the H3-kana-H4 described in SEQ ID NO: 4, as described in Example 1, integrate SEQ ID NO: 4 into the fadE gene position of the genome of BW25113 that lacks the fabH gene so that the fadE gene is deleted and integrated SEQ ID NO: 4 IDNO: 4 to delete the KanR resistance gene.

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Abstract

The present invention provides an olivetol synthase variant and an engineered microorganism expressing it for the production of olivetol and olivetolic acid, more specifically an engineered Escherichia coli, wherein the engineered Escherichia coli is modified to express olive The alcohol synthase variant is characterized in that the olive alcohol synthase variant comprises one or more mutations selected from the following mutations compared with its wild type: I303T, I52L, S56A, H262M and K263R. The engineered E. coli achieves improved yields of olivetol and olivetolic acid.

Description

technical field [0001] The invention relates to the field of microorganisms and enzymes, in particular to olive alcohol synthase (OLS) variants and engineered microorganisms expressing the same for producing olivetol (olivetol, OL) and olivetolic acid (olivetolic acid, OA). Background technique [0002] Cannabinoids are derived from the plant cannabis (cannabinoids) Cannabis sativa ), a large class of chemical molecules with more than 150 species. Cannabinoids currently used internationally include cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabigerol (CBG). CBD is one of the main chemical components in plant cannabis and is a non-addictive component of cannabinoids. THC is the main psychoactive substance in marijuana, which can be addictive and is strictly controlled in various countries around the world. Because of its low content in plant cannabis, CBG is usually classified as a trace cannabinoid or a rare cannabinoid. Because it is the common precursor of oth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/54C12N15/53C12R1/19
CPCC12N15/52C12N9/88C12N9/1029C12N15/70C12Y404/01026C12Y203/01086C12Y203/01206C12P7/22C12P7/42C12N9/001C12Y203/0118Y02E50/10
Inventor 杜德尧王高艳张倩宗朕李明月邱悦悦尹进张浩千
Owner BLUEPHA CO LTD
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