Limonene-producing yarrowia lipolytica and construction method and application thereof

A technology of Yarrowia lipolytica and limonene, applied in the field of molecular biology, can solve the problem of low yield of limonene heterogeneous synthesis, and achieve the effect of wide application prospects

Inactive Publication Date: 2018-09-28
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a Yarrowia lipolytica genetic engineering strain that introduces D-limonene synthase gene (DLS) or L-limonene synthase gene (LLS) in view of the low yield of limonene heterologous s...

Method used

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  • Limonene-producing yarrowia lipolytica and construction method and application thereof
  • Limonene-producing yarrowia lipolytica and construction method and application thereof
  • Limonene-producing yarrowia lipolytica and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of Yarrowia lipolytica strain producing limonene

[0052] The construction process of recombinant strains is as follows: image 3 shown.

[0053] According to the nucleotide sequence of D-limonene synthase gene in Genebank (210bp truncated at the 5' end and 262bp truncated at the 3' end, and the start codon ATG and the stop codon TGA were added before and after) and L-limonene synthase The nucleotide sequence of the gene (196bp truncated at the 5' end and 345bp truncated at the 3' end and the start codon ATG and the stop codon TGA were added before and after) respectively according to the codon usage preference of Yarrowia lipolytica Optimize and add a His tag at the 3' end of the gene, and then synthesize it by a gene synthesis company. The nucleotide sequence of the synthesized D-limonene synthase gene is shown in SEQ ID NO.1, and the nucleotide sequence of the L-limonene synthase gene is shown in Shown in SEQ ID NO.2. According to the synth...

Embodiment 2

[0067] Embodiment 2: Fermentative production of limonene by genetically engineered bacteria obtained in embodiment 1

[0068] Each of the engineering bacteria Po1g ku70Δ-DHR and Po1g ku70Δ-LHR obtained in Example 1 and the host strain Yarrowia lipolytica Po1g ku70Δ were each taken one ring, inoculated in a 250mL Erlenmeyer flask containing 25mL of YPD medium, 30°C, 225rpm / min, after 24 hours of shaking culture, inoculate 1% of the inoculum in a 250mL Erlenmeyer flask containing 25mL of YPD medium, 30°C, 225rpm / min, continue shaking for 16 hours, then inoculate 1% of the inoculum into 25mL of YPD medium 250mL Erlenmeyer flask based on base, and add 10% dodecane, 28 ℃, 225rpm / min, shaking flask fermentation for 5 days.

[0069] After the fermentation, all the fermentation broth was poured into a 50mL centrifuge tube, centrifuged at 7500rpm, 4°C for 5min, and the organic phase was taken to pass through the membrane, and the gas chromatography-mass spectrometry was used for testi...

Embodiment 3

[0073] Embodiment 3: Fermentative production of limonene by genetically engineered bacteria obtained in embodiment 1

[0074] Take one ring each of the engineering bacteria Po1g ku70Δ-DHR and Po1g ku70Δ-LHR obtained in Example 1, inoculate them in a 250mL Erlenmeyer flask containing 50mL of YPD medium, at 28°C, 250rpm / min, shake and cultivate for 24h, then inoculate with 1 % of the inoculum was inoculated in a 250mL Erlenmeyer flask containing 50mL of YPD medium, at 28°C, 250rpm / min, continued shaking for 16 hours, and then inoculated into a 250mL Erlenmeyer flask containing 50mL of YPD medium at an inoculum of 1%, and added 8% dodecane, 30°C, 200rpm / min, shake flask fermentation for 4 days.

[0075] After inspection, the output of heterologously synthesized limonene by the engineering strain producing D-limonene in the middle is 0.6175mg / fermentation broth, and the output of heterologously synthesizing limonene by the engineering strain producing L-limonene is 0.3564mg / L ferm...

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Abstract

The invention belongs to the technical field of molecular biology, and relates to limonene-producing yarrowia lipolytica genetic engineering bacterium and the application thereof. The genetic engineering bacterium is obtained by introducing a D-limonene synthase gene or an L-limonene synthase gene into a yarrowia lipolytica host and overexpressing a 3-hydroxy-3-methyl glutaryl-coenzyme A reductase(HMGR) gene. After shake flask fermentation in an YPD culture medium, limonene can be heterologously synthesized, the yield of D-limonene heterologously synthesized by a D-limonene-producing engineering strain is 0.6305mg/L and the yield of L-limonene heterologously synthesized by an L-limonene-producing engineering strain is 0.3672mg/L.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology, and relates to a Yarrowia lipolytica genetically engineered bacterium producing limonene and an application thereof. Background technique: [0002] Yarrowia lipolytica is a typical representative unconventional yeast. The yeast is non-pathogenic, the maximum growth temperature is generally below 34°C, and has been identified as a GRAS (generally regarded as safe) microorganism. Different from Saccharomyces cerevisiae, this yeast is strictly aerobic and has the characteristics of dimorphic growth. Growth conditions such as carbon source, nitrogen source, and pH will affect the colony morphology of this bacteria, so it has become a research tool for yeast and fungal hyphae. Differentiated model strains. Another remarkable advantage of this yeast is that it widely exists in various foods and various living environments, because it can utilize a wide range of carbon sources, including or...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/66C12P5/00C12R1/645
CPCC12N9/88C12N15/66C12N15/815C12P5/00C12Y402/03016C12Y402/0302
Inventor 于爱群庞亚如赵雅坤张翠英肖冬光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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