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Recombinant saccharomycetes for high yield of sandalwood oil as well as construction method and application thereof

A construction method and technology of sandalwood oil, applied in the field of genetic engineering, can solve problems such as no successful cases

Active Publication Date: 2020-07-21
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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  • Recombinant saccharomycetes for high yield of sandalwood oil as well as construction method and application thereof
  • Recombinant saccharomycetes for high yield of sandalwood oil as well as construction method and application thereof
  • Recombinant saccharomycetes for high yield of sandalwood oil as well as construction method and application thereof

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Experimental program
Comparison scheme
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] Embodiment 2 fermentation recombinant strain produces sandalwood oil

[0079] The high-yielding sandalwood oil-producing recombinant Saccharomyces cerevisiae strain constructed in this study adopts synthetic medium fermentation conditions and uses 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 ammoniu...

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 recombinant saccharomycetes for high yield of sandalwood oil as well as a construction method and application of the recombinant saccharomycetes. The method comprises the following steps: 1) integrating genes such as acetoacetyl-CoA thiolase gene and the like which are derived from saccharomyces cerevisiae and are driven by a strong promoter and CYP450 reductase gene whichis driven by a weak promoter into a saccharomyces cerevisiae BY4742 genome of an original strain in a homologous recombination manner; 2) replacing a promoter of a squalene synthase gene in the recombinant bacteria; knocking out genes such as galactose modulin 80 genes; and (3) inserting a sandalene synthase gene driven by a strong promoter and a CYP450 monooxygenase gene into the multi-copy freeplasmid to obtain an expression plasmid, and introducing the expression plasmid into the recombinant saccharomyces cerevisiae obtained in the step (2) to obtain the recombinant saccharomyces cerevisiae for high yield of sandalwood oil. According to the method, the defect that sandalwood oil is extracted from sandalwood is overcome, and the yield of each liter of sandalwood oil of the recombinant yeast strain reaches 1g. Industrial production requirements can be met.

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 Applications(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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