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Yeast engineering bacterium for producing d-limonene and construction method thereof

A yeast engineering and construction method technology, applied in the field of genetic engineering, to achieve good application prospects, simple construction method, and reduced feedback inhibition

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 d -Limonene has not been reported 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 d - limonene synthase sequence, after codon optimization, the whole gene synthesis is carried out; artificially synthesized d -The limonene synthase gene was integrated into the plasmid pRS304 to construct an integrated expression vector. After transforming Escherichia coli DH5a, the transformants were selected, the plasmids were extracted and subjected to BamHI and SpeI After digestion, a 1.8kb band appeared, which proved that the integrated expression vector had been constructed successfully.

Embodiment 2

[0037] Example 2 Construction of Yeast Engineering 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 ). 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 the pH to 5.6, sterilize at 115°C for 20 min), pick the transformant that grows normally on the plate after transformation, extract the genome PCR verification, a 1.8kb band appears, and the control fails to PCR the same band, proving successful integration to the genome.

example 3

[0039] Example 3 Fermentative production of limonene

[0040] Seed medium (g / L): 20 g of glucose, 1.7 g of YNB and 5 g of ammonium sulfate, 0.2 g of leucine, 0.2 g of histidine, 0.2 g of uracil, pH 5.6. Add 20 agar when making slopes and plates, and sterilize at 115 °C for 20 min.

[0041]Fermentation medium (g / L): 20 g of glucose, 1.7 g of YNB and 5 g of ammonium sulfate, 0.4 g of leucine, 0.4 g of histidine, 0.4 g of uracil, pH 5.6. Add 20 agar when making slopes and plates, and sterilize at 115 °C for 20 min.

[0042] 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 48-56 h, shake the flask to ferment at 30°C, 200rpm, d - Limonene yield was 6 mg / L.

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PUM

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Abstract

The invention discloses a yeast engineering bacterium for producing d-limonene and a construction method thereof, belonging to the field of gene engineering. In the invention, limonene synthetase from Citrusunshiu is heterologously expressed in Saccharomyces cerevisiae CEN.PK2-1C by metabolic engineering modification to increase ways (mevalonic acid (MVA) carbon metabolic flux) for synthesizing terpenoids, thereby obtaining a yeast engineering bacterium strain CEN.PK2 limonene for producing d-limonene at high yield. Compared with the original strain CEN.PK2-1C, the modified yeast engineering bacterium can produce d-limonene by metabolism, the feedback inhibition on d-limonene is obviously lowered, and the yield of d-limonene can reach 13 mg / L, thus the invention has wide application prospects.

Description

technical field [0001] The present invention relates to a high yield d - Limonene yeast engineering bacteria and its construction method, especially a molecular means to modify the terpene production (MVA) pathway and heterologous introduction d - limonene synthase, thereby regulating metabolic flux achieved d - Method for excessive accumulation of limonene, belonging to the field of genetic engineering. Background technique [0002] Limonene, also known as Ningene. Limonene is a chain terpene alcohol, with α- and β-isomers, and L-( d ), dextrorotation ( l ) two photoisomers. In different sources, it is mostly a mixture of isomers. The racemate exists in Clareba oil, jasmine oil, or chemically synthesized limonene; the L-form d - Limonene is present in essential oils such as linoleum, sassafras, bergamot, lavender, rosewood, etc. [0003] Limonene is an important spice, used in floral flavors, perfumes, soaps and aroma industries, etc. It is the most frequently use...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/52C12P5/00
Inventor 陈坚刘继栋孙明雪周景文堵国成
Owner JIANGNAN UNIV
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