Phaeodactylum tricornutum bifunctional enzyme and application thereof

A bifunctional enzyme and Phaeodactylum technology, applied in the field of genetic engineering and metabolic engineering, to reduce useless workload and increase oil content

Inactive Publication Date: 2017-10-27
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its lipid content is still far from the development of the production pr...

Method used

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  • Phaeodactylum tricornutum bifunctional enzyme and application thereof
  • Phaeodactylum tricornutum bifunctional enzyme and application thereof
  • Phaeodactylum tricornutum bifunctional enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment -1

[0041] Cloning of PtWS / DGAT Gene from Phaeodactylum tricornutum

[0042]Using the DGAT gene sequence of Thalassiosira pseudonana as the starting sequence, through the NCBI database Blast search, it was found that there was an enzyme in Phaeodactylum tricornutum (XP_002184474.1) with two partially overlapping conserved functional regions, namely, wax ester synthase and two Fatty glycerol acyltransferase domain. According to the gene sequence (such as SEQ ID No: 2, without intron structure) encoding the enzyme, the primers are designed as follows:

[0043] Pri-forward1:5'-ATGGATGTCTTTGGCAGC-3'

[0044] Pri-reverse1:5'-AAGTACGACTGTAAATT-3'

[0045] Under sterile conditions, cultivate Phaeodactylum tricornutum with seawater culture solution f / 2 to the logarithmic growth phase, the temperature is 23°C, the light is 4000-6000LUX, and the light-dark ratio is 12h / 12h. Take 500ml algae liquid and centrifuge at 9000rpm at 4°C for 5min. Quickly put the algae into liquid nitrogen to f...

Embodiment 2

[0049] Expression of Phaeodactylum tricornutum PtWS / DGAT in Saccharomyces cerevisiae

[0050] Using the correctly sequenced pMD-18T plasmid containing PtWS / DGAT in Example 1 as a template, primer Pri-forward2:5'-AAGCTTAGGATGGATGTCTTTGGC-3'

[0051] and Pri-reverse2: 5'-GGATCCTTAGAAGTACGACTGTAA-3'

[0052] Clone the PtWS / DGAT gene, and add restriction sites HindIII and BamHI to its 5' end, respectively. The PtWS / DGAT was inserted into the vector pYES2 by double enzyme digestion and ligation reaction, named pYES2-PtWS / DGAT.

[0053] Three types of yeast culture and screening media are pre-configured: A. Conventional medium YPD medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, autoclaved; B. Screening medium SC-Ura culture Base: 0.67% yeast medium nitrogen source (Yeast nitrogen base (YNB)), 2% glucose, 0.074% Do Supplement (-Ura), low pressure sterilization; C. induction medium: 0.67% Yeast nitrogen base (YNB), 2% galactose, 1% raffinose, 0.074% DoSupplement (-Ura), ...

Embodiment 3

[0070] Expression of Phaeodactylum tricornutum PtWS / DGAT in Escherichia coli

[0071] Using the correctly sequenced pMD-18T plasmid containing PtWS / DGAT in Example 1 as a template, primer Pri-forward3:5'-CATATGATGGATGTCTTTGGC-3'

[0072]and Pri-reverse3:5'-CTCGAGTTAGAAGTACGACTGTAA-3'

[0073] Clone the PtWS / DGAT gene, and add restriction sites NdeI and XhoI at its 5' end and 3' end. The PtWS / DGAT was inserted into the vector pET28a by double enzyme digestion and ligation reaction, named pET28a-PtWS / DGAT.

[0074] Using the 42°C heat shock method, pET28a-PtWS / DGAT was introduced into E. coli BL21(DE3). After screening with kanamycin, a resistant mutant was obtained and named BL21-PtWS / DGAT.

[0075] Prepare the antibiotics and inducers used in advance. Dissolve 260mg of kanamycin in 5.2mL sterile water, pass through a 0.22μm filter membrane to sterilize (autoclaved) and dispense into EP tubes (autoclaved) to make a 50mg / ml mother solution, -20 Store at ℃. Dilute 1g of IPT...

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Abstract

The invention relates to the field of genetic engineering and metabolic engineering and in particular to a phaeodactylum tricornutum bifunctional enzyme (wax ester synthetase/diacylglycerol glycerinum acyltransferase) and application thereof. The phaeodactylum tricornutum bifunctional enzyme is wax ester synthetase/diacylglycerol glycerinum acyltransferase, the amino acid residue of the enzyme is as shown in SEQ ID No:1 in a sequence table, or has 90% or greater of homology of an amino acid as shown in SEQ ID No:1 in the sequence table, and is capable of encoding sequences of proteins of same functions. The phaeodactylum tricornutum bifunctional enzyme, namely the wax ester synthetase/diacylglycerol glycerinum acyltransferase can be applied to metabolic engineering research of microalgae, is capable of increasing the content of lipid, and has wide application prospects in development of biodiesel. Genes of the wax ester synthetase/diacylglycerol glycerinum acyltransferase are over-expressed in phaeodactylum tricornutum, mutant strains of which the grease content is remarkably increased are acquired, and the enzyme can be applied to biodiesel production processes.

Description

technical field [0001] The invention relates to the fields of genetic engineering and metabolic engineering, in particular to a bifunctional enzyme of Phaeodactylum tricornutum (wax ester synthase / diacylglycerol acyltransferase) and its application. Background technique [0002] With the continuous development of global economic integration, the world's energy situation is becoming increasingly tense, and the extensive use of petroleum fuels has made the energy crisis and environmental problems increasingly prominent. Finding a sustainable new energy source has become a concern of all countries. As a green and sustainable new energy source, biodiesel has received more and more attention. my country's industrialized biodiesel is currently mainly prepared from oil crops such as soybean and rapeseed, oil tree fruits such as oil palm and jatropha, animal fats, and waste cooking oil. Although the raw materials are diverse, the sources are very limited. Even if they are added tog...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P7/64C12N1/13C12R1/89
CPCC12N9/1029C12P7/64C12P7/649C12Y203/01075C12Y203/01158Y02E50/10
Inventor 崔玉琳秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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