Yarrowia lipolytica capable of producing bisabolene and constructing method for yarrowia lipolytica and use of yarrowia lipolytica

A technology of Yarrowia lipolytica and bisabolene, applied in the field of molecular biology, can solve the problem of low yield of bisabolene heterologous synthesis

Inactive Publication Date: 2020-05-01
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to provide a strain introducing α-bisabolene synthase gene or β-bisabolene synthase gene or γ-bisabolene synthase gene or γ-bisabolene Yarrowia lipolytica genetically engineered bacteria with bisabolene synthase gene and the use of the strain in heterologous synthesis of bisabolene, which can synthesize bisabolene and construct bisabolene by means of metabolic engineering or synthetic biology. Engineering strains for alkene production (refer to attached figure 2 )

Method used

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  • Yarrowia lipolytica capable of producing bisabolene and constructing method for yarrowia lipolytica and use of yarrowia lipolytica
  • Yarrowia lipolytica capable of producing bisabolene and constructing method for yarrowia lipolytica and use of yarrowia lipolytica
  • Yarrowia lipolytica capable of producing bisabolene and constructing method for yarrowia lipolytica and use of yarrowia lipolytica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Construction of a Yarrowia lipolytica strain producing bisabolene

[0077] According to the nucleotide sequence of α-bisabolene synthase gene, nucleotide sequence of β-bisabolene synthase gene and nucleotide sequence of γ-bisabolene synthase gene in Genebank, lipolysis Saccharomyces pylori codon usage preference was respectively optimized for codons and a His tag was added to the 3' end of the gene, and then synthesized by a gene synthesis company. The nucleotide sequence of the synthesized α-bisabolene synthase gene is shown in the sequence listing as SEQ ID Shown in NO: 1; the nucleotide sequence of the synthetic β-bisabolene synthase gene is shown in SEQ ID NO: 2 in the sequence listing; the nucleotide sequence of the synthetic γ-bisabolene synthase gene is shown in sequence Shown in SEQID NO: 3 in the list. According to the gene sequence after synthesis, the HMGR reference sequence and the integrated plasmid sequence in Genebank, each primer (shown in Ta...

Embodiment 2

[0090] Example 2: Comparison of Bisabolene Fermentation Production by Genetic Engineering Bacteria

[0091] (1) Experimental method

[0092] Take one loop of the engineering bacteria and the starting strain Yarrowia lipolytica Po1gΔku70, inoculate in a 250mL Erlenmeyer flask containing 25mL YPD medium, 30°C, 225r / min, vibrate for 24 hours, inoculate with 1% inoculum in a 25mL Erlenmeyer flask containing In a 250mL Erlenmeyer flask of YPD medium, 30°C, 225r / min, continue shaking culture for 16h, then inoculate into a 250mL Erlenmeyer flask containing 50mL of YPD medium at an inoculum size of 1%, and add 10% Twelve alkanes, 28°C, 225r / min, shake flask fermentation for 5 days.

[0093] Composition of YPD medium: peptone 20g / L, yeast extract powder 10g / L, glucose 20g / L, the balance is water, pH5.7-5.8, 115°C, sterilized for 20min.

[0094] After the fermentation, all the fermentation broth was poured into a 50mL centrifuge tube, centrifuged at 7500rpm, 4°C for 5min, the organic ...

Embodiment 3

[0114] Example 3: Fermentative production of bisabolene by genetically engineered bacteria using an optimized cheap carbon source

[0115] The engineering bacteria Po1gΔku70α-HR, Po1gΔku70β-HR and Po1gΔku70γ-HR obtained in Example 1 and the starting strain Po1gΔku70 of Yarrowia lipolytica get a circle, inoculate in the YPD medium that contains 25mL, according to the above-mentioned embodiment 2 (1) Fermentation culture according to the fermentation method, 30°C, 225r / min, shaking culture for 24h, inoculate 1% inoculum in a 250mL Erlenmeyer flask containing 25mL YPD medium, 30°C, 225r / min, continue shaking culture for 16h Inoculate 1% of the inoculum into a 250mL Erlenmeyer flask containing 50mL of optimized cheap medium, add 10% dodecane, 28°C, 225r / min, and shake the flask for 5 days of fermentation.

[0116] The composition of the cheap optimized medium is: peptone 20g / L, yeast extract powder 10g / L, kitchen waste oil 11.8mL / L, magnesium sulfate 2g / L, the balance is water, pH...

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Abstract

The invention discloses genetically engineered bacteria of yarrowia lipolytica capable of producing bisabolene and use of the genetically engineered bacteria, and belongs to the technical field of molecular biology. The genetically engineered bacteria are obtained by introducing an alpha-bisabolene synthase gene, a beta-bisabolene synthase gene or a gamma-bisabolene synthase gene into a host of the yarrowia lipolytica and by overexpression of a 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (HMGR gene). After the genetically engineered bacterium undergo shake-flask fermentation in respective yeast extract peptone dextrose (YPD) mediums, the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 alpha-HR is 100.22 mg / L, the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 beta-HR is 5.66 mg / L, and the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 gamma-HR is 3.55 mg / L. After themediums are optimized, the yield of the bisabolene produced by the genetically engineered bacteria Po1g Delta ku70 alpha-HR is 162.24 mg / L, the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 beta-HR is 20.811 mg / L, and the yield of the bisabolene produced by genetically engineered bacteria Po1g Delta ku70 gamma-HR is 6.25 mg / L.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a Yarrowia lipolytica genetic engineering bacterium producing bisabolene and its application. 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 (generally regarded as safe, GRAS) safety-level microorganisms. Different from Saccharomyces cerevisiae, this yeast is strictly aerobic, and the maximum biomass value it can achieve is relatively large. Another remarkable advantage of Yarrowia lipolytica is that it widely exists in various foods and various living environments, because the yeast can use a wide range of carbon sources, including organic acids, alkanes, alkenes, oils, Alcohols, esters and other cheap carbon sources. In addition, Yarrowia lipolytica can produce and (or) secrete various metabolites during ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P5/00C12R1/645
CPCC12N9/0006C12N9/88C12N15/815C12P5/002C12Y101/01088C12Y402/03038C12Y402/0304C12Y402/03055
Inventor 于爱群赵雅坤李建李圣龙赵禹朱坤张翠英肖冬光
Owner TIANJIN UNIV OF SCI & TECH
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