Brewer's yeast engineering bacterium for producing valencene and construction method and application of brewer's yeast engineering bacterim

A technology of Saccharomyces cerevisiae and a construction method, applied in the biological field, can solve the problems of intricate plant metabolic engineering, low growth rate, unstable yield, etc., and achieve the effects of improving metabolic flux, efficient production, and improving cell productivity

Active Publication Date: 2019-08-13
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But plant metabolic engineering is intricate and requires many efforts
A more promising approach is to obtain products through plant cell culture. This approach is currently used to produc...

Method used

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  • Brewer's yeast engineering bacterium for producing valencene and construction method and application of brewer's yeast engineering bacterim
  • Brewer's yeast engineering bacterium for producing valencene and construction method and application of brewer's yeast engineering bacterim
  • Brewer's yeast engineering bacterium for producing valencene and construction method and application of brewer's yeast engineering bacterim

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0069] Example 2 Construction of guide RNA (gRNA) recombinant expression vector

[0070] Using the gRNA plasmid p426-SNR52p-gRNA.CAN1.Y-SUP4t (purchased from Addgene) targeting canavanine transportase as the backbone to construct the gRNA recombinant expression vector p426-SNR52p targeting knockout of ROX1 and downregulation of ERG9 -gRNA.ROX1-ERG9.Y-SUP4t. The specific implementation is as follows:

[0071] 1.PCR amplification of rox1-gRNA, erg9-gRNA expression vector fragments

[0072] Using p426-SNR52p-gRNA.CAN1.Y-SUP4t as a template, ROX1-1 and ROX1-2 (ROX1-1 and ROX1- 2 respectively have two fragments (nucleotide sequences shown in SEQ ID NO: 36 and SEQ ID NO: 37).

[0073] Using p426-SNR52p-gRNA.CAN1.Y-SUP4t as a template, Tong-F / ERG9-R, ERG9-F / Tong-R primer pairs amplified respectively to obtain ERG9-1 and ERG9-2 (ERG9-1 and ERG9- 2 respectively have two fragments (nucleotide sequences shown in SEQ ID NO: 38 and SEQ ID NO: 39).

[0074] 2. Construction of p426-SNR5...

Embodiment 3

[0092] Example 3 Construction of Overexpression tHMG1 Expression Vector

[0093] With YEp181-P constructed in Example 1 TDH3 -VS-T ADH1 As the starting vector, the complete tHMG1 expression cassette was introduced to obtain a recombinant expression vector for overexpressing tHMG1, labeled as YEp181-tHMG1-VS1. The specific implementation is as follows:

[0094] 1. Using the genome of Saccharomyces cerevisiae CEN.PK2-1Ca (same as Example 1) as a template, using tHMG1-U / tHMG1-D as amplifying primers to amplify tHMG1 with restriction sites BamHI and SmaI Gene fragment.

[0095] 2. Construction of recombinant expression vector for overexpressing tHMG1

[0096] Digest YEp181-P with BamHI and SmaI TDH3 -VS-T ADH1 The vector and tHMG1 fragments, the vector is recovered and purified by conventional gel cutting, the vector and tHMG1 fragments are ligated and transformed to obtain a recombinant expression vector that overexpresses tHMG1, labeled as YEp181-P TDH3 -tHMG1-T ADH1 . ...

Embodiment 4

[0103] Embodiment 4 produces the construction of valenciene engineering bacteria

[0104] The gRNA expression vector p426-SNR52p-gRNA.ROX1-ERG9.Y-SUP4t constructed in Example 2 was transferred into yeast competent cells, and realized with the assistance of the p414-TEF1p-Cas9-CYC1t vector (purchased from Addgene Company) Site-directed editing of the yeast genome. Then, the recombinant expression vectors constructed in Examples 1 and 3 were transformed into yeast competent cells subjected to site-directed editing to obtain recombinant yeast expression strains, which were respectively marked as PK2-1, PK2-2, PK2-3 and PK2-4. The specific implementation is as follows:

[0105] 1. Amplify homologous recombination fragments

[0106] The primer pair ROX1-M-F / ROX1-M-R and UAS-M-F / UAS-M-R were respectively used as templates through the annealing extension between the upstream and downstream, and the homologous recombination fragments donor1 and donor2 were respectively obtained.

...

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Abstract

The invention discloses a brewer's yeast engineering bacterium for producing valencene and a construction method and application of the brewer's yeast engineering bacterium. Overall inhibiting factorgenes in a yeast genome are subjected to knockout, a squalene synthetase gene is subjected to down reduction of expression, valencene synthetase is expressed, HMG-CoA reductase is overexpressed, the promoter of the valencene synthetase is optimized, and therefore the valencene is obtained. The key gene in the metabolic pathway is overexpressed through a free vector, the metabolic flux of the valencene in the synthetic route is increased, and the yeast cell factory is optimized; in combination with the suitability research of the promoter of the valencene synthetase, the brewer's yeast engineering bacterium obtained through culture optimization achieves efficient production of the valencene, and the dry cell weight yield of the valencene can reach 124.40 mg/g; the brewer's yeast engineeringbacterium has a wide industrial application prospect and positive economic and social development significance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Saccharomyces cerevisiae engineering bacterium producing valenciene and its construction method and application. Background technique [0002] Valenciene, light yellow clear liquid, has the characteristic aroma of sweet orange oil, miscible with absolute ethanol, insoluble in water, relative density (20°C) 0.9194g / L, refractive index 1.5001, flash point 100°C, Molecular formula is C 15 h 24 . Valenciene, an important sesquiterpenoid, has been a key factor in consumer preferences for a wide range of products, including food, beverages, fragrances, personal care and home care products. [0003] Valenciene is currently mainly obtained from citrus essential oils through distillation, extraction and other processes, which are cumbersome and costly. Citrus essential oils are obtained by cold-pressing the peels of citrus fruits. It takes 2.5 million kilograms of oranges to...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P5/00C12R1/865
CPCC12N15/81C12P5/005C12N9/1085C12Y205/01021C12N9/0006C12Y101/01034C12N9/00
Inventor 李爽欧阳小丹察亚平朱晁谊
Owner SOUTH CHINA UNIV OF TECH
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