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Yarrowia lipolytica genetically engineered bacterium for producing linalool and application of Yarrowialipolytica genetically engineered bacterium

A technology of Yarrowia lipolytica and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problem of low yield and the like, and achieve the effects of simple operation, good prospects, stable and reliable reaction

Pending Publication Date: 2018-12-14
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally introduce linalool synthase gene biofermentation method to produce linalool by exogenous source, but introduce exogenous linalool synthase gene in Saccharomyces cerevisiae at present, its output is not high, only 0.095mg / L (Pegah Amiri et al. al., Metabolic engineering of Saccharomyces cerevisiae for linalool production, Biotechnology Letters, March 2016, Volume 38, Issue 3, pp 503–508) or 0.127mg / L (Sun Mingxue, etc., regulating the isoprenoid synthesis pathway of Saccharomyces cerevisiae to strengthen linalool Synthesis, Chinese Journal of Bioengineering, June 23, 2013, 29(6): 751-759), there is an urgent need for genetically engineered bacteria with high production of linalool

Method used

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  • Yarrowia lipolytica genetically engineered bacterium for producing linalool and application of Yarrowialipolytica genetically engineered bacterium
  • Yarrowia lipolytica genetically engineered bacterium for producing linalool and application of Yarrowialipolytica genetically engineered bacterium
  • Yarrowia lipolytica genetically engineered bacterium for producing linalool and application of Yarrowialipolytica genetically engineered bacterium

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preparation example Construction

[0035] For the preparation method of plasmid pINA1312, see Nicaud, J.M., Madzak, C., Broek, P., Gysler, C., Duboc, P., Niederberger, P., Gaillardin, C., 2002, Protein expression and secretion in the yeast Yarrowia lipolytica . FEMS yeast research 2, 371-379.

[0036] For the preparation method of plasmid pINA1269, see Madzak C, Tréton B and Roland SB. Stronghybrid promoters and integrative expression / secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowialipolytica. J Mol Microbiol Biotechnol.(2000)2(2):207 -216.

[0037] 质粒p1269-HMG1、p1269-IDI1、p1269-IDI1IDI1、p1269-IDI1IDI1IDI1、p1269-HMG1ERG8、p1269-HMG1ERG10、p1269-HMG1ERG12,p1269-HMG1ERG19、and p1269-HMG1IDI1的制备方法参见本申请人CN201610817882.X的专利申请 (hereinafter referred to as the patent); and Cao X, LvYB, Chen J, Imanaka T, Wei LJ, & Hua Q Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction. Biotechnology for biofuels. (2016) 9:214 (hereinafter referred t...

Embodiment 1

[0038] Example 1 Construction of Engineering Bacteria Strain CXY01

[0039] The whole gene was synthesized by Shanghai Jierui Bioengineering Co., Ltd. and constructed into p1312 plasmid. In the present invention, the single-fragment seamless cloning kit is used for the construction of the plasmid, and the supplier is Nanjing Novizan Biotechnology Co., Ltd.

[0040] (1) Whole gene synthesis of the optimized kiwifruit gene LIS (its nucleotide sequence is shown in the sequence table SEQ ID No.1); the LIS gene and the plasmid p1312 are digested by the restriction site PmlI, and the LIS is connected to the plasmid p1312 , to obtain plasmid p1312-LIS.

[0041] (2) Linearize the plasmid pINA1312-LIS obtained in step (1) with the enzyme NotI (the restriction endonucleases used in the present invention were all purchased from Takara Bao Biology), and transform it into Yarrowia lipolytica Po1f by homologous recombination Among them, the initial strain CXY01 capable of producing linalo...

Embodiment 2

[0042] Embodiment 2 constructs engineering bacteria strain CXY21-24

[0043] For the specific construction method of the plasmid, please refer to the patent and literature, which are briefly described as follows:

[0044] (1) Using the Yarrowia lipolytica genome as a template, HMG1 (the accession number in NCBI is GB: YALI0E04807g, and the primer sequences are shown in the literature as P7 and P8), IDI1 (the accession number in NCBI is YALI0F04015g) were amplified respectively. , the primer sequences are shown in the literature as P9 and P10).

[0045] (2) Link the genes obtained in the above (1) and Example 2 to the plasmid p1269 through the restriction site PmlI to obtain plasmids p1269-HMG1, p1269-IDI1; p1269-IDI1IDI1 and p1269-IDI1IDI1IDI1 specific construction method See above reference.

[0046] The plasmid obtained in step (2) was linearized with the restriction endonuclease BsrGI, and transformed into the initial strain CXY01 obtained in Example 1 by homologous recom...

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Abstract

The invention discloses a Yarrowia lipolytica genetically engineered bacterium for producing linalool and application of the Yarrowia lipolytica genetically engineered bacterium. The genetically engineered bacterium is formed by the following steps: optimizing a linalool synthetase gene LIS from Actinidia arguta and transforming the optimized linalool synthetase gene LIS into Yarrowia lipolytica Polf; and on the basis, over expressing one or more of endogenous genes HMG1, IDI1 or ERG synchronously. Additionally, a carbon source such as glucose, glycerin, fructose, citric acid and pyruvic acidadded when in fermentation of the genetically engineered bacterium and the concentration of the carbon source are selected, so that the yield of linalool is improved further. The highest yield of linalool can be 6.96mg / L, the content of linalool in a unit cell is 939.04 [mu]g / g DCW (dry cell weight), and the yield is extremely high. The genetically engineered bacterium can be used for large-scalecommercialized production and is good in prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a linalool-producing Yarrowia lipolytica genetically engineered bacterium and an application thereof. Background technique [0002] The structure of linalool contains two isoprenoid groups and ten carbon atoms, and it has been highly concerned as an aromatic substance in essential oils and a food additive, as well as advanced bioenergy. Linalool, as a ring-opening monoterpene tertiary alcohol, is one of the main components of many essential oils. About 70% of the terpenoids in floral fragrances are represented by linalool. Linalool is added to shampoos, soaps, shower gels and other cosmetics and household detergents as an aroma and flavor substance, as well as processed foods and beverages. In addition, linalool is also a key precursor for the production of products such as vitamin E, microbial A, farnesol, citronellol and ionone. Linalool also has antifungal, ba...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N9/88C12N15/81C12P7/04C12R1/645
CPCC12N1/16C12N9/88C12N15/81C12P7/04C12Y402/03
Inventor 花强韦柳静曹选林嘉宇
Owner EAST CHINA UNIV OF SCI & TECH
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