Yarrowia lipolytica engineering bacterium for producing triacetate lactone by using xylose and application of yarrowia lipolytica engineering bacterium

A technology of Yarrowia lipolytica and engineering bacteria, applied in application, genetic engineering, fermentation and other directions, to achieve the effect of optimizing the fermentation process

Active Publication Date: 2021-09-17
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the method of constructing xylose metabolism pathway in Yarrowia lipolytica to heterologously synthesize triacetin by genetic engineering method has not been reported yet.

Method used

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  • Yarrowia lipolytica engineering bacterium for producing triacetate lactone by using xylose and application of yarrowia lipolytica engineering bacterium
  • Yarrowia lipolytica engineering bacterium for producing triacetate lactone by using xylose and application of yarrowia lipolytica engineering bacterium
  • Yarrowia lipolytica engineering bacterium for producing triacetate lactone by using xylose and application of yarrowia lipolytica engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Mechanism research based on glycerol as xylose metabolism "response switch"

[0044]In order to explore the regulatory mechanism of glycerol in the process of xylose metabolism in Yarrowia lipolytica, Yarrowia lipolytica po1f containing the empty plasmid PYLXP' was cultured in a leucine-deficient xylose fermentation medium (xylose 40g / L , YNB1.7g / L, ammonium sulfate 1.1g / L, CSM-Leu 0.69g / L, the rest is water) in a 250mL shake flask, add 3‰ pure glycerol at the same time, 30℃, 250rmp / min, culture to logarithm The bacteria were extracted during the growth period, and qPCR was used to determine the intracellular xylose reductase gene yl.xyl1, xylitol dehydrogenase gene yl.xyl2, xylitol dehydrogenase gene yl.xyl2, Expression level of xylulokinase gene yl.xyl3.

[0045] β-actin was selected as an internal reference gene, and the relative mRNA expression levels of these three genes were determined. See the test results figure 2 , showing that compared with t...

Embodiment 2

[0048] Example 2: Construction of a synthetic pathway for producing triacetolactone from xylose

[0049] In order to further enhance the ability of Yarrowia lipolytica to metabolize xylose to produce high-value chemical triacetolactone, the optimized 2-pyrone synthase gene gh2ps and xylose reductase gene yl were introduced into Yarrowia lipolytica po1f .xyl1, xylitol dehydrogenase gene yl.xyl2, and xylulokinase gene yl.xyl3 expression cassettes to obtain Yarrowia lipolytica recombinant strain Yali01, the gene sequence of which is shown in SEQ ID NO.1 to SEQ ID NO.4 Show.

[0050] (1) Plasmid construction

[0051] The optimized gh2ps gene is synthesized by the company, and yl.xyl1, yl.xyl2, and yl.xyl3 are all from the Yarrowia lipolytica po1f genome. Amplified with gh2ps-F / gh2ps-R, yl.xyl1-F / yl.xyl1-R, yl.xyl2-F / yl.xyl2-R, yl.xyl3-F / yl.xyl3-R as primers respectively Optimized nucleotide sequences gh2ps (SEQ ID NO.1), yl.xyl1 (SEQ ID NO.2), yl.xyl2 (SEQ ID NO.3) yl.xyl3 gene...

Embodiment 3

[0067] Embodiment 3: Yarrowia lipolytica recombinant strain Yali01 fermentation produces triacetin

[0068] (1) Culture of recombinant bacteria

[0069] First, the recombinant bacterium Yali01 obtained in Example 2 was connected to 3mL leucine-deficient seed medium (xylose 20g / L; YNB1.7g / L; ammonium sulfate 5g / L; CSM-Leu0.69g / L, The rest is water.) in a test tube at 30°C, cultivated in a shaker at 250rpm for 2-3 days, inoculated into a 250mL Erlenmeyer flask containing 30mL of leucine-deficient fermentation medium by 2% inoculum size, and placed in a shaker Cultured in Li for 7 days, the shaker conditions were set at 30°C, 250rpm, and samples were taken for analysis every 24h.

[0070] The components and contents of the fermentation medium are as follows: 40g / L xylose, 1.7g / L YNB, 1.1g / L ammonium sulfate, 0.69g / LCSM-LEU, 3g / L glycerol, and the rest is water.

[0071] (2) Detection of fermentation broth

[0072] Use a microplate reader or a UV spectrophotometer to measure th...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant yarrowia lipolytica strain for producing triacetate lactone by using xylose and application of the recombinant yarrowia lipolytica strain. According to the engineering bacterium, yarrowia lipolytica is taken as an original strain, a 2-pyrone synthetase coding gene gh2ps is introduced, and meanwhile, a xylose reductase coding gene, a xylitol dehydrogenase coding gene and a xylulokinase coding gene are overexpressed, so that the recombinant yarrowia lipolytica engineering bacterium is obtained. According to the method, the fermentation process is further optimized, the optimal production process for producing the triacetate lactone from the xylose by using the yarrowia lipolytica is developed, the yield of the yarrowia lipolytica reaches the highest level in a shake flask reported at present and ranges from 3g / L to 6g / L, and a foundation is laid for providing a chassis strain for subsequently developing a cheap carbon source to produce a high-value platform compound, namely the triacetate lactone.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Yarrowia lipolytica strain for producing triacetolactone by utilizing xylose and application thereof. Background technique: [0002] Triacetolactone is a "bridge compound" linking biotransformation and chemical catalysis. It contains a 2-pyrone structure and can be converted into various high-value intermediates or end products by hydrolysis, catalytic hydrogenation, etc., such as acetyl Acetone, γ-caprolactone, thus identified as a potential biorenewable platform chemical, is a valuable precursor for the synthesis of commercial compounds and is in great demand in traditional fields such as materials and chemicals. In addition, because triacetolactone is non-toxic, harmless and low-corrosion, it is also widely used in food, medicine and other life and health fields. [0003] At present, the production methods of triacetin mainly include chemical synthesis ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/54C12N15/81C12P17/06C12R1/645
CPCC12N9/0006C12N9/1205C12N9/1037C12N15/815C12P17/06C12Y101/01307C12Y101/01009C12Y207/01017C12Y203/01074C12N2800/102
Inventor 邓利黄晓斓刘欢王芳
Owner BEIJING UNIV OF CHEM TECH
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