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Preparation method and application of high-yield 2-phenylethanol with candida glycerinogenes engineering bacterium

A Candida and phenylethanol technology, applied in the field of genetic engineering, can solve the problems of difficult product purification, large loss, toxicity hindering the production of 2-phenylethanol, etc., and achieve the effect of overcoming the toxic effect and increasing the yield

Inactive Publication Date: 2017-11-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

2-Phenylethyl alcohol mainly exists in rose, jasmine, narcissus and other plant essential oils in nature, and its natural content is low. Now it is usually extracted by steam distillation, but the loss is relatively large, which often cannot meet people's needs.
[0003] The chemical synthesis of 2-phenylethanol has many disadvantages, such as endangering human health and environmental safety, and difficulty in product purification.
Therefore, the toxicity of 2-phenylethanol to bacteria has largely hindered the strategy of improving the production of 2-phenylethanol through metabolic modification.

Method used

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  • Preparation method and application of high-yield 2-phenylethanol with candida glycerinogenes engineering bacterium
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  • Preparation method and application of high-yield 2-phenylethanol with candida glycerinogenes engineering bacterium

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Experimental program
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Effect test

Embodiment 1

[0021] Construction of heterozygous mutants with single-copy knockout of ALD gene

[0022] (1) Synthesis of knockout fragments

[0023] A 2.5kb ALD3 gene fragment was amplified by PCR, which included the nucleic acid sequence of 500bp-0bp upstream of ALD3 gene (PloALDa, SEQ ID No. 2), the nucleic acid sequence of 0p-500bp downstream of ALD3 gene and ALD3 gene (TloALDa, SEQ ID No. 2) .3). And connect with pMD19-T simple to obtain recombinant plasmid 19T-ALDa. Then, using 19T-Ald3a as a template, reverse PCR was used to obtain a PloALDa-19T-TloALDa fragment containing two homology arms, and the fragment of the selection marker HisG-URA5-HisG (HUH) was ligated through the restriction site to obtain the knockout plasmid 19T-PloALDa -HUH-TloALDa, named plasmid pADHuha, and the plasmid pADHuha was cut with restriction endonuclease BamH I to obtain a linearized knockout fragment

[0024] (2) Knockout of the first chain of ALD3 gene

[0025] Pick a ring of Candida glycerol-produci...

Embodiment 2

[0029] Construction of homozygous mutants with double-copy knockout of ALD3 gene

[0030] (1) Synthesis of knockout fragments

[0031] A 1.5kb ALD3 gene fragment was amplified by PCR and ligated with pMD19-T simple to obtain the recombinant plasmid 19T-ALD3b. Then, using 19T-Ald3b as a template, reverse PCR was used to obtain a PloALDb-19T-TloALDb fragment containing two homology arms, which was ligated with the screening marker HisG-URA5-HisG (HUH) fragment through the restriction enzyme site to obtain the knockout plasmid 19T-PloALDb -HUH-TloALDb, named plasmid pADHuhb, and the plasmid pADHuhb was cut with restriction endonuclease BamH I to obtain a linearized knockout fragment.

[0032] (2) Knockout of the second chain of ALD3 gene

[0033] A heterozygous mutant strain with a single-copy knockout of the ALD3 gene was picked and inoculated in liquid YPD medium and cultured at 30°C overnight. Take 100ul of the overnight culture medium and transfer it to fresh YPD, and incu...

Embodiment 3

[0035] Pick 1 ring of original Candida glyceride and ALD3 gene double-copy knockout homozygous mutant strains and insert them into the seed medium (yeast powder 10g / L, peptone 20g / L, glucose 20g / L, the balance is water ), under the conditions of 30 ° C, 200 r / min, shaking culture for 18 h, to obtain liquid seeds. The obtained liquid seeds are inserted into the inoculum containing 30mL fermentation medium (L-phenylalanine 7g / L, glucose 90g / L, KH 2 PO 4 5g / L, yeast powder 1g / L, MgSO 4 ·7H 2(00.5g / L, the balance is water) in a 250mL conical flask, the fermentation temperature is controlled to be 30°C, the rotational speed is 200r / min, the time is 72h, and the fermentation is completed.

[0036] The determination method of 2-phenylethanol in the fermentation broth is analyzed by high performance liquid chromatography (HPLC), and the details are as follows: the fermentation broth is centrifuged at 10,000 r / min, and then filtered with a 0.45 μm microporous membrane after centrif...

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Abstract

The invention discloses a preparation method and application of a glycerol-producing Candida engineering bacterium with high yield of 2-phenylethyl alcohol, belonging to the technical field of genetic engineering. Candida glycerinogenes CCTCC M 93018 is a diploid strain. Compared with haploid strains, it has the advantages of fast growth, strong viability, stable genetics, and prevention of lethal or harmful mutations. At the same time, Candida glycerologenus is an excellent 2-phenylethanol producing strain, and its ability to tolerate high concentrations of 2-phenylethanol (4g / L) provides a favorable basis for the metabolic transformation of the strain. The invention utilizes the gene knockout technique to knock out the acetaldehyde dehydrogenase gene ALD3 of the diploid glycerol-producing Candida strain 2-phenethyl alcohol synthesis competition pathway. Compared with the original glycerologenic Candida strain, the ALD3 knockout mutant strain increased the production of 2‑phenylethanol by 18.5%, reaching 3.65 g / L.

Description

technical field [0001] The invention relates to a preparation method and application of a high-yield 2-phenylethanol-producing Candida glyceride engineering bacterium, and belongs to the technical field of genetic engineering. Background technique [0002] 2-Phenylethanol has an elegant, delicate and long-lasting rose aroma. It is the base of most flavors and is highly valued in the flavor and fragrance industry. In recent years, 2-Phenylethanol has been widely used in industries such as medicine, food, cosmetics, tobacco and daily chemical products, and the market demand is increasing. 2-Phenylethanol mainly exists in natural plant essential oils such as rose, jasmine, narcissus, etc., and the natural content is relatively low. Now it is commonly extracted by steam distillation, but the loss is large, which often cannot meet people's needs. [0003] The chemical synthesis of 2-phenylethanol has many disadvantages, such as endangering human health and environmental safety, ...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/81C12P7/22C12R1/72
CPCC12P7/22C12N9/0008C12N15/815C12Y102/0101C12Y102/99003C12Y102/99007
Inventor 王小婉诸葛斌王玉芹陆信曜宗红
Owner JIANGNAN UNIV
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