Recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol and construction method and application thereof

A technology of Yarrowia lipolytica and Yarrowia lipolytica, which is applied in the field of bioengineering, can solve the problems of cumbersome chemical synthesis steps, abnormally complex structure, and unsustainability, and achieve enhanced homologous recombination capabilities, improved genetic stability, and constructed effective method

Active Publication Date: 2019-06-21
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, (-)-α-bisabolol is mainly obtained by chemical synthesis and plant extraction. Due to the extremely complex structure of (-)-α-bisabolol, its chemical synthesis steps are cumbersome and follow-up Difficult to separate, currently it is mainly obtained by distilling Eucalyptus brasiliensis
However, due to limited resources, the number of Brazilian eucalyptus is decreasing sharply, so the method of distilling and extracting (-)-α-bisabolol using Brazilian eucalyptus not only cannot meet people's needs, but also is unsustainable

Method used

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  • Recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol and construction method and application thereof
  • Recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol and construction method and application thereof
  • Recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1, the amplification of gene element and the preparation of target plasmid

[0033] (1) Preparation of target gene

[0034] According to the nucleotide sequence of the (-)-α-bisabolol synthase coding gene MrBBS provided by NCBI, after codon optimization, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the optimized (-)-α- Bisabolol synthase encoding gene OptBBS was inserted into plasmid pUC57 to obtain plasmid pUC57-OptBBS. The nucleotide sequence of OptBBS is shown in SEQ ID NO.1.

[0035] According to the nucleotide sequence (M37309.1) of the 3(β)-isopropylmalate dehydrogenase coding gene leu in Yarrowia lipolytica provided by NCBI, Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize leu, and 3( β)-Isopropylmalate dehydrogenase encoding gene expression cassette was inserted into plasmid pUC57 to obtain plasmid pUC57-leu.

[0036] According to the nucleotide sequence (genebank accession number AJ306421.1) and hisG tag (gene...

Embodiment 2

[0086] The construction of embodiment 2 recombinant bacteria

[0087] (1) Construction of Recombinant Bacteria 1

[0088] (1) The plasmid pUC-leu-A08-OptBBS containing the OptBBS gene expression cassette was introduced into Yarrowialipolytica Po1fΔku70, and the OptBBS expression cassette was integrated into the A08 site of the genome to obtain recombinant bacterium 1. The specific method is as follows: (1) Yarrowia lipolytica Po1fΔku70 was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) to prepare competent cells. (2) Transform pUC-leu-A08-OptBBS into Yarrowia lipolytica Po1fΔku70 using the Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation for homologous recombination. (3) The screening medium SD-Leu was used to screen, and the positive clone identified by PCR was named recombinant bacteria 1. The screening medium SD-Leu contains: glucose 20g / L, YeastNitrogen Base (YNB, yeast nitrogen base ammonium sul...

Embodiment 3

[0124] Embodiment 3, the application of recombinant bacteria 1-8 in the production of (-)-α-bisabolol

[0125] 1. Cultivation of engineering bacteria and product extraction

[0126] The recombinant bacteria 1-8 in Example 2 were used to produce (-)-α-bisabolol respectively. The specific method is as follows: activate the recombinant bacteria, culture in YPD liquid medium at 30° C. and 220 rpm for 16 hours to obtain seed liquid. The seed solution was inoculated in 50 ml of fermentation medium with an inoculum size of 1%, and cultured with shaking at 30° C. and 220 rpm for 1 day, then added n-dodecane with 10% of the volume of the fermentation liquid, and continued shaking for 3 days. After the fermentation, the fermentation broth was transferred to a 50ml centrifuge tube, centrifuged at 5000rpm for 15min, and the organic phase was collected for later use.

[0127] Wherein the fermentation medium contains 55g / L glucose, 10g / L yeast extract and 20g / L tryptone. 2. Qualitative a...

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Abstract

The invention provides recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol and a construction method and application thereof and relates to the field of biological engineering. The recombinant Yarrowia lipolytica to produce (-)-alpha-bisabolol is constructed by inserting an (-)-alpha-bisabolol synthase, farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl CoA synthase expression kit into the genome of Yarrowia lipolytica. The recombinant Yarrowia lipolytica is suitable for efficiently synthesizing (-)-alpha-bisabolol; the construction method is efficient and is simple to perform.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a recombinant Yarrowia lipolytica for producing (-)-α-bisabolol and its construction method and application. Background technique [0002] (-)-α-Bisabolol is an unsaturated sesquiterpene alcohol mainly found in the essential oils of Brazilian eucalyptus (Eremanthuserythropappus) and German chamomile (Matricaria recutita). (-)-α-Bisabolol has been widely used in oral hygiene products and skin care products due to its good anti-inflammatory, antibacterial, antiseptic and moisturizing properties, and has been added to big brands such as Estee Lauder and Clinique in skin care products. The U.S. Food and Drug Administration (FDA) has approved (-)-α-bisabolol as a safe ingredient to be safely added to various daily chemical products such as mouthwash, skin care products, and shower gels (Waltimo T et al. al., Arch Oral Biol. 2013, 58:10-6). [0003] At present, (-)-α-bisabolol is mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/60C12N15/54C12N15/53C12P7/22
Inventor 纪晓俊马一榕王凯峰丁颖王维鉴黄和
Owner NANJING UNIV OF TECH
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