High-yield s-linalool genetic engineering strain and building method thereof

A technology of genetically engineered bacteria and construction methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, fungi, etc., to achieve the effects of simple construction methods, reduced feedback inhibition, and good application prospects

Inactive Publication Date: 2011-05-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Metabolic engineering for Saccharomyces cerevisiae production s- Linalool has no related reports in China

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0035] Example 1 Construction of expression vector

[0036] According to NCBI published s- The sequence of linalool synthase is codon-optimized for whole gene synthesis; artificially synthesized s- The linalool synthase gene was integrated into the integrated plasmid pRS304 to construct an integrated expression vector. After transforming Escherichia coli DH5a, the transformants were selected, the plasmids were extracted and subjected to EcoRI and SpeI After digestion, a 1.9 kb band appeared, proving that the integrated expression vector had been successfully constructed.

Embodiment 2

[0037] Embodiment 2 Construction of genetically engineered bacteria

[0038] The constructed expression plasmid was transformed into Saccharomyces cerevisiae CEN.PK2-1C ( MATa ; ura3 -52; trp1 -289; leu2 -3,112; his3 D 1; MAL2-8 C ; SUC2 ), said bacterial strain was purchased from German EUROSCARF strain collection center. Since the recombinant plasmid carries trp1 Gene, transformed Saccharomyces cerevisiae CEN.PK2-1C competent, spread to YNB containing histidine, leucine, uracil (glucose 20 g / L, YNB 6.7 g / L, solid medium plus 20 g / L Agar, adjust pH 5.6, sterilize at 115°C for 20 min), pick the transformants that grow normally on the plate after transformation, extract the genome PCR verification, a 1.9kb band appears, and the control fails to PCR the same band, which proves the successful integration into on the genome.

example 3

[0039] Example 3 Production of Linalool by Fermentation

[0040] Inoculate engineered yeast bacteria from the slant into 20 mL of seed medium, place on a shaker at 30°C with a rotation speed of 200 rpm, cultivate for 20-24 h to the mid-log phase, and transfer to the fermentation at a transfer rate of 10%. In the culture medium, ferment and cultivate for 40-48 h, shake the flask to ferment at 30°C, 200rpm, s- The yield of linalool was 5 mg / L.

[0041]Seed medium (g / L): 20 g glucose, 1.7 g YNB and 5 g ammonium sulfate, 0.2 g leucine, 0.2 g histidine, 0.2 g uracil, pH 5.6. Add 20 g of agar when making slopes and plates, and sterilize at 115 °C for 20 min.

[0042] Fermentation medium (g / L): 20 g glucose, 1.7 g YNB and 5 g ammonium sulfate, 0.4 g leucine, 0.4 g histidine, 0.4 g uracil, pH 5.6. Add 20 g of agar when making slopes and plates, and sterilize at 115 °C for 20 min.

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PUM

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Abstract

The invention discloses a high-yield s-linalool genetic engineering strain and a building method, which belong to the field of genetic engineering. Through the metabolic engineering reformation, linalool synthetase from actinidiaarguta is expressed in saccharomyces cerevisiae CEN.PK2-1 in a heterologous way, the terpene synthesis path-mevalonic acid (MVA) path carbon metabolizing flow is increased, and the high-yield s-linalool genetic engineering strain CEN. PK2 linalool is obtained. Compared with an initial strain CEN. PK2-1C, the modified genetic engineering strain CEN. PK2 linalool can metabolize and produce s-linalool, in addition, the feedback inhibition of the s-linalool is obviously reduced, the yield of the s-linalool can reach 12 mg / L, and the genetic engineering strain has good application prospects.

Description

technical field [0001] The present invention relates to a product s- Linalool yeast engineering bacteria and its construction method, especially a molecular means to modify the terpene production (MVA) pathway and heterologous introduction s- Linalool synthase, which regulates metabolic flux to achieve s- A method for excessive accumulation of linalool belongs to the field of genetic engineering. Background technique [0002] Linalool (linalool), also known as linalool, coriander alcohol, scientific name 3,7-dimethyl-1,6 octadien-3-ol. Linalool is a chain terpene alcohol, with α- and β-isomers, and L-( s ), dextrorotation ( l ) two photoisomers. In different sources, it is mostly a mixture of isomers. The racemate is present in Clara oil, jasmine oil, or chemically synthesized linalool; the L-form s- Linalool is found in essential oils such as linoleum, sassafras, bergamot, lavender, and rosewood. [0003] Linalool is an important spice, used in floral flavors, perfu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/52C12P7/02C12R1/865
Inventor 陈坚刘继栋孙明雪周景文堵国成
Owner JIANGNAN UNIV
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