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High-yield heavy oil aureobasidium pullulans strain as well as construction method and application thereof

A technology of Aureobasidium pullulans and construction method, which can be applied to other methods of inserting foreign genetic materials, botanical equipment and methods, microorganism-based methods, etc., and can solve problems such as low capacity of fermentation to produce heavy oil.

Active Publication Date: 2020-01-21
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that Aureobasidium pullulans has low ability to ferment and produce heavy oil when xylose is used as carbon source, and to provide a recombinant strain of Aureobasidium pullulans and its construction method for improving heavy oil production

Method used

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  • High-yield heavy oil aureobasidium pullulans strain as well as construction method and application thereof
  • High-yield heavy oil aureobasidium pullulans strain as well as construction method and application thereof
  • High-yield heavy oil aureobasidium pullulans strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of the Aureobasidium pullulans recombinant strain that improves the ability to produce heavy oil

[0043] The starting strain used in this example is Aureobasidium pullulans P30. The Escherichia coli DH5α was purchased from Takara Company. The YPD medium is a general complete medium, and the solid medium contains 2% imported agar powder.

[0044] Based on the Aureobasidium pullulans genome data and the integrated plasmid sequence, the following primers were designed. The underline indicates the KpnI restriction site.

[0045] The primers used in the present embodiment 1 of table 1

[0046]

[0047]

[0048] The PCR amplification system used in the present embodiment 1 of table 2

[0049]

[0050]

[0051] The PCR amplification system used in the present embodiment 1 of table 3

[0052]

[0053] (1) Construction of recombinant plasmid pUC-HPT

[0054] The construction process of the recombinant plasmid pUC-HPT is as follows...

Embodiment 2

[0059] Embodiment 2: Recombinant bacterial strain uses xylose as carbon source fermentation experiment

[0060] Pick the strains and inoculate them in the seed culture medium, culture them with shaking at 28°C and 240r / min for 24h to obtain the seed liquid; insert the seed liquid into the fermentation medium with an inoculum size of 6%, and carry out shake flask fermentation. The fermentation conditions are: 28°C, 240r / min shaking culture, and the fermentation ended after 7 days.

[0061] Among them, the composition of the seed medium is: xylose 20g / L, yeast extract powder 1.0g / L, K 2 HPO 4 4.0g / L, (NH 4 ) 2 SO 4 0.8g / L, MgSO 4 0.2g / L, NaCl 4.0g / L, balance water, pH 6.0, sterilized at 121°C for 20min.

[0062] The composition of the fermentation medium is: xylose 50g / L, yeast extract powder 2.0g / L, KNO 3 0.8g / L, NaCl 2.0g / L, K 2 HPO 4 5.0g / L, MgSO 4 0.3g / L, the balance is water, pH 5.0, sterilized at 121°C for 20min.

[0063] Fermentation experiment of Aureobasi...

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Abstract

The invention discloses a high-yield heavy oil aureobasidium pullulans strain as well as a construction method and application thereof and belongs to the field of genetic engineering. By knocking outa pullulan synthetase gene and overexpressing an ACL (adenosine triphosphate-citrate lyase) gene, the yield of heavy oil of screened aureobasidium pullulans can be increased obviously. After 7 days offermentation with xylose as a carbon source, the yield of heavy oil of a recombinant strain is up to 19.4372g / L, the yield of heavy oil of a parent strain is 10.0325g / L, and the yield of heavy oil ofthe recombinant strain is increased by 93.74% when being compared with that of the heavy oil of the parent strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering, specifically relates to the synthesis of polyol lipids (Liamocins), in particular to the overexpression of ATP-citrate lyase (ACL) gene to increase the yield of Aureobasidium pullulans heavy oil, and the heavy oil content has been greatly improved. Background technique [0002] Aureobasidium pullulans (Aureobasidium pullulans) is a kind of polymorphic saprophytic fungi with a very complex life cycle, belonging to the phylum Deuterobacteria, the order Aureobasidium pullulans, and the genus Aureobasidium pullulans. During its growth and development, there are yeast-like cells, budding spores, swollen spores, chlamydospores and mycelial cells. [0003] Heavy oil is a kind of microbial oil, which is an important secondary metabolite of Aureobasidium pullulans. It is denser than water and generally deposited at the bottom of the fermentation broth. Heavy oils are divided into alkanes and polyalcoho...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/90C12N15/60C12P7/64C12R1/645
CPCC12N9/88C12N15/80C12N15/902C12P7/6463C12Y401/03C12N9/1025C12Y203/03008C12R2001/645C12N1/145C12P7/62C12P19/44
Inventor 陈叶福黄思瑶郑鹏王远花郭建郭学武肖冬光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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