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Construction method and application of engineering escherichia coli for producing humulone

A technology of Escherichia coli and a construction method, applied in the field of humulones, can solve the problems of large consumption of organic solvents, unstable product quality, strong dependence on raw materials, etc., achieve stable product quality, get rid of dependence on hop raw materials, and retain biological active effect

Active Publication Date: 2020-09-15
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention proposes a construction method of engineering Escherichia coli for the production of humulones and its application in the production of humulones, aiming to solve the existing problems in the method of extracting humulones from hops in the prior art with many operating steps and organic solvents. Large consumption, low purity, strong dependence on raw materials and unstable quality of different batches of products

Method used

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  • Construction method and application of engineering escherichia coli for producing humulone
  • Construction method and application of engineering escherichia coli for producing humulone
  • Construction method and application of engineering escherichia coli for producing humulone

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Embodiment 1

[0042] A method for constructing engineering Escherichia coli for producing humulones of the present invention comprises the following steps:

[0043] Preparation of S1 Competent Cells

[0044] The construction of engineering strains requires competent cells that can absorb DNA molecules in the surrounding environment. The preparation method of competent cells is as follows:

[0045] a. First, you need to take out the preserved BL21(DE3)-Trc-low strain from the -80°C ultra-low temperature freezer, place the glycerol tube containing the BL21(DE3)-Trc-low strain on ice to thaw slightly, and a small amount of it will melt After the bacterial liquid was collected, operate it in an ultra-clean bench, use a sterilized 10 μL pipette tip or an inoculation loop to streak onto a non-resistant LB solid plate, and incubate in a water-proof constant temperature incubator at 37°C for about 12 to 14 hours (BL21(DE3)-Trc-low strain is relatively slow in growth and reproduction compared with ...

Embodiment 2

[0065] The application of a kind of engineering escherichia coli of the present invention in the biosynthesis of humulone comprises the following steps:

[0066] S1 Trc-low engineering strain fermentation experiment

[0067] The fermentation experiment of Trc-low engineering strains started with the successful transformation of pACYCDuet-1-CCL2-VPS-PT2 plasmid and pTrcHis 2B-IDI-PT1-Monooxygenase plasmid. The transformed strains were resistant to chloramphenicol and ampicillin It takes about 14-16 hours for a single colony to grow on the flat plate. The steps of the Trc-low engineering strain fermentation experiment are as follows:

[0068] (1) Preparation of primary seed solution

[0069] This operation is carried out in a clean bench. First, draw 10 mL of LB liquid from the Erlenmeyer flask containing sterilized LB liquid medium into a 50 mL sterilized dry centrifuge tube, then add 7 μL of chloramphenicol and 7 μL of For ampicillin, shake gently to mix antibiotics and medi...

Embodiment 3

[0085] On the basis of embodiment two, change the parameter of shake flask fermentation, in shake flask fermentation process, fermentation medium is: (NH 2 ) 4 SO 4 1g / L, K 2 HPO 4 ·3H 2 O 3g / L, KCl 1.7g / L, sodium citrate 1g / L, betaine 1g / L, glucose 20g / L, citric acid 1g / L, yeast extract 10g / L; sterilize by high pressure steam at 115℃ for 30min;

[0086] First, add 2mL / L magnesium sulfate mother solution and 1mL / L trace element mother solution to the fermentation medium; then, add 0.7mL / L ampicillin mother solution and 0.7mL / L chloramphenicol mother solution; finally, add 7.5mL / L formazan Valonic acid and 15mL / L leucine mother liquor, mevalonate and leucine mother liquor are added to the fermentation broth after the inducer is added, and mevalonate is added to the fermentation broth in 3 batches;

[0087] The concentration of the magnesium sulfate mother liquor is 0.24g / L, and the trace element mother liquor is composed of the following components: ammonium molybdate tet...

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Abstract

The invention provides a construction method and application of engineering escherichia coli for producing humulone. The construction method comprises the following steps: taking engineering escherichia coli as an original strain, constructing a gene obtained after evolution of prenyl pyrophosphate isomerase derived from the engineering escherichia coli, a gene obtained after evolution of prenyl transferase derived from humulus lupulus and a monooxygenase gene into a vector plasmid to obtain a first expression vector; constructing a gene obtained after evolution of pentanedione synthase derived from humulus lupulus, a gene obtained after evolution of cytoplasmic coenzyme A ligase and an gene segment obtained after evolution of prenyl transferase into another vector plasmid to obtain a second expression vector; transferring the expression vectors into engineering escherichia coli to obtain engineering escherichia coli for producing humulone. The invention also provides a method for synthesizing humulone by using the engineering escherichia coli biological method. The synthesis method provided by the invention has the advantages of few steps, high purity and stable quality, gets ridof dependence on humulus lupulus raw materials, and avoids massive consumption of solvents.

Description

technical field [0001] The invention relates to the technical field of humulones, in particular to a method for constructing engineering Escherichia coli for producing humulones and its application in producing humulones. Background technique [0002] Humulone belongs to a kind of picric acid in hops, which is a kind of α-acid, and is the direct precursor source of bitterness in the beer brewing process, that is, the precursor of iso-α-acid that provides the main bitterness in beer. Alpha-acids are part of the soft resin, consisting of dipglutarylated phloroglucinol derivatives with variable acyl side chains. Humulone is a phloroglucinol derivative isolated from the glands of hops. Its English name is humulone. Its structure has the common characteristics of natural products and is relatively complex. It consists of more carbon molecules. 21 h 30 o 5 It is the chemical molecular formula of humulone, the specific molecular weight is 362.466, and the CAS number of humulone ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/26C07C49/743C07C45/79C07C45/78C12R1/19
CPCC12N15/70C12N9/90C12N9/1085C12N9/0004C12N9/00C12Y503/03002C12Y205/01C12Y113/1105C07C49/743C07C45/79C07C45/786C07C2601/16Y02A50/30
Inventor 邹慧斌黄静玲张楠王亚群梁秀红
Owner QINGDAO UNIV OF SCI & TECH
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