A kind of recombinant yeast with high sandalwood oil production and its construction method and application

A construction method and technology of recombinant Saccharomyces cerevisiae, applied in the field of high-yielding sandalwood oil-producing yeast strains and its construction, can solve problems such as no successful cases

Active Publication Date: 2021-06-18
TIANJIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cross-species heterologous expression of the enzymes required to catalyze the synthesis of such small molecules has many technical barriers. At present, there is no successful case of microbial fermentation industrial production of sandalwood oil.

Method used

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  • A kind of recombinant yeast with high sandalwood oil production and its construction method and application
  • A kind of recombinant yeast with high sandalwood oil production and its construction method and application
  • A kind of recombinant yeast with high sandalwood oil production and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0034] The construction method of the recombinant saccharomyces cerevisiae of embodiment 1 high yield sandalwood oil

[0035] The invention describes a method for constructing a recombinant Saccharomyces cerevisiae with high sandalwood oil production. The method includes a large number of molecular biology experimental techniques, including restriction endonuclease digestion of DNA molecules, connection of DNA fragments, and colonization of DNA molecules. Bacillus transformation, cultivation of Escherichia coli, extraction of Escherichia coli plasmid, transformation of Saccharomyces cerevisiae, etc. Unless otherwise specified, the specific technical details are conventional means known to those skilled in the art. Escherichia coli and plasmid pUC19 used in the gene cloning work were purchased from Beijing Quanshijin Biotechnology Co., Ltd., item number: CD101-02. Plasmid pEASY-Blunt Simple was purchased from Beijing Quanshijin Biotechnology Co., Ltd., hereinafter referred to ...

Embodiment 2

[0078] Example 2 Fermentation of recombinant strains to produce sandalwood oil

[0079] The high-yield sandalwood oil-producing recombinant Saccharomyces cerevisiae strains constructed in this study adopt synthetic medium fermentation conditions and use bioreactor fermentation to produce sandalwood oil. The media formulations involved are as follows:

[0080] The components of the plate medium include: 6.7g / L yeast base without amino acid nitrogen source, 1.92g / L yeast synthetic medium without uracil supplement, 20g / L glucose, 2.0% agar powder. Among them, yeast without amino acid nitrogen source base, manufacturer name: Yeast Nitrogen Base Without Amino Acids, brand: SIGMA-ALDRICH, item number: V900895-100G. Yeast Synthetic Drop-out Medium Supplements without uracil: manufacturer name Yeast Synthetic Drop-out Medium Supplements without uracil, brand: SIGMA-ALDRICH, product number: Y1501-20G.

[0081] The components of the seed medium include: 19.5g / L glucose, 15g / L ammonium...

Embodiment 3

[0091] GC-MS detection of embodiment 3 high-yield sandalwood oil strains and the quantification of its sandalwood oil output

[0092] (1) GC-MS detection method of samples

[0093] Sandalwood oil sample processing: After the organic phase of the fermentation broth is filtered through a 0.22 μm filter membrane, it can be directly sent for testing. When quantifying, it should be properly diluted with n-dodecane as a solvent according to the estimated concentration, and then placed in a sample bottle for delivery. Sample detection was used to ensure that the detection concentration was within the linear range of the standard curve during quantification. During the GC-MS quantification process, the internal standard was selected from α-Humulene purchased from Sigma-Aldrich. The reference product was commercially purchased sandalwood oil, which was used as a qualitative and quantitative reference after purification. GC-MS adopts Shimadzu QP2010GC-MS detection system, ion scanning ...

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Abstract

The invention discloses a recombinant yeast with high sandalwood oil production and its construction method and application. The method is as follows: 1) using homologous recombination to drive the acetoacetyl-CoA thiolase gene derived from Saccharomyces cerevisiae with a strong promoter Isogenic genes and CYP450 reductase genes driven by weak promoters were integrated into the genome of the starting strain Saccharomyces cerevisiae BY4742; 2) replacing the promoter of the squalene synthase gene in the recombinant bacteria; knocking out the galactose regulatory protein 80 gene and other genes; 3 ) Insert the santalene synthase gene driven by a strong promoter and the CYP450 monooxygenase gene into the multi-copy episomal plasmid to obtain an expression plasmid, and introduce it into the recombinant Saccharomyces cerevisiae obtained in step (2) to obtain high-production sandalwood oil Recombinant Saccharomyces cerevisiae. The invention overcomes the deficiency that the production of sandalwood oil is all extracted from sandalwood, and the output of the recombinant yeast strain can reach 1g per liter of sandalwood oil. Meet industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a high-yield sandalwood oil-producing yeast strain optimized through genetic engineering transformation and its construction method and application. Background technique [0002] Sandalwood oil belongs to sesquiterpenes, mainly exists in the core material of sandalwood, and is a widely used spice. The main component of sandalwood oil is a mixture of four alcohols, namely α-santalol (α-santalol), α-exo-bergamotol, epi-β-santalol and β-santalol (β-santalol). The quality of sesame oil is the content of santalol (Baldovini N, Delasalle C, Joulain D.Phytochemistry of the heartwood fromfragrant Santalum species: a review[J].Flavour and Fragrance Journal,2011,26(1):7-26.), Sandalwood oil is widely used in the field of cosmetics, perfume and aroma health care because of its strong fragrance (Page T, Southwell I, Russell M, et al. Geographic and Phenotypic Variation in Heartwood and Esse...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N1/19C12P7/02C12R1/865
CPCC12N15/81C12P7/02
Inventor 张雁张立新胡逸灵
Owner TIANJIN UNIV
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