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Chlamydomonas reinhardtii hemolytic phosphatidic acid acyltransferase and its gene and application

An acyltransferase, hemolytic technology, applied in the field of molecular biology and biology, can solve problems such as unclear LPAAT, and achieve the effect of low viscosity and low ignition point

Active Publication Date: 2020-06-16
GUOTOU BIO TECH INVESTMENT CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, it is not clear whether LPAAT derived from different organelles in lower plant algae has similar preferences. If there is a similar preference, it can be used to regulate and control the fatty acid content in biological cells (especially algal cells). Composition structure, so that C16 or C18 fatty acids constitute the main component of total fatty acids in cells, will largely solve the technical problems mentioned above, and make oil-producing biological cells (especially microalgae) truly become an ideal source of biodiesel

Method used

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  • Chlamydomonas reinhardtii hemolytic phosphatidic acid acyltransferase and its gene and application
  • Chlamydomonas reinhardtii hemolytic phosphatidic acid acyltransferase and its gene and application
  • Chlamydomonas reinhardtii hemolytic phosphatidic acid acyltransferase and its gene and application

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Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1 gene cloning and sequence feature analysis

[0048] The CrLPAAT1 gene provided by the present invention is based on the Cre09.g398289.t1.1 gene published by the JGI Chlamydomonas reinhardtii genome database (http: / / phytozome.jgi.doe.gov / pz / portal.html) as a reference gene, and the design is specific Primer (forward primer, as shown in SEQ ID No.3, 5'-ATGGCGCGTAAAAGCAGTTTGGCTC-3', reverse primer, as shown in SEQ ID No.4, 5'-CTGCTCATCCGGCGCCATCTCTGT-3'), using Chlamydomonas reinhardtii cDNA The library (the construction of the cDNA library is prepared and stored according to the "Molecular Cloning Experiment Guide" by conventional methods) is used as a template, and the cDNA sequence is obtained by PCR amplification, such as figure 1 shown. The PCR reaction system was: 2 μl template DNA, 10 μl 2×GoTaq DNA polymerasemix (purchased from Promega (Beijing) Biotechnology Co., Ltd.), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), and finally supplemente...

Embodiment 2

[0051] Example 2 Function Verification

[0052] Using Chlamydomonas reinhardtii cDNA as a template, primers (forward primer, shown in SEQ ID No.5, 5'-tgacggatccATGGCGCGTAAAAGCAGTTTGGCTC-3', reverse primer, As shown in SEQ ID No.6, 5'-actgaagcttTCACTGCTCATCCGGCGCCA TCTCTGT-3') and high-fidelity polymerase pfu were amplified by PCR to obtain 5' and 3' open reading frame DNA fragments containing corresponding restriction sites. T4 DNA ligase was used to connect the fragment with the double-digested expression vector pQE30 to obtain a recombinant plasmid. The recombinant plasmid was transformed into LPAAT-deficient temperature-sensitive Escherichia coli SM2-1 (plsC-) (SM2-1 was obtained from Yale University Escherichia Coli Resource Center CGSC) competent cells by heat shock method. Compared with the control group (containing the pQE30 empty vector), the SM2-1 strain transformed with the CrLPAAT1 gene could survive at 42°C after the recombinant protein was induced by IPTG, while ...

Embodiment 3

[0054] Example 3 In vitro enzyme activity test

[0055] In the present invention, the SM2-1 Escherichia coli transformed with the CrLPAAT1 gene and the SM2-1 Escherichia coli transformed with the pQE30 empty plasmid were cultured at 30° C. and 0.5 mM IPTG was added to induce protein expression for 6 hours, and the cells were collected by centrifugation. The membrane fraction was extracted by mechanical crushing and gradient centrifugation and used as a crude enzyme for in vitro enzyme activity experiments. In the experiment, a variety of acyl-CoAs with different carbon chain lengths and degrees of saturation were selected as acyl donors, and the optimal substrate was determined by observing the accumulation of the product phosphatidic acid (PA). The conditions of the enzyme reaction are: 250 μM lysophosphatidic acid (LPA), 200 μM acyl-CoA (Acyl-CoA), 1 mM MgCl in 200 μl 100 mM potassium phosphate buffer (pH 7.4) 2 with 20 μg crude enzyme. After the crude enzyme was added, th...

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Abstract

The invention discloses Chlamydomonas reinhardti lysophosphatidic acid acyltransferase, and a gene and application thereof. The Chlamydomonas reinhardti lysophosphatidic acid acyltransferase is 1) a protein composed of amino acids as shown in SEQ ID No. 1; or 2) a protein derived from the protein 1) by subjecting the amino acid sequence as shown in SEQ ID No. 1 to substitution, deletion or addition of one or plural amino acids and having same activity as the protein 1). Research results show that the Chlamydomonas reinhardti lysophosphatidic acid acyltransferase has intense selectivity on 16:0-CoA. Through excess expression of the gene, a ratio of C16:0 aliphatic acid in biological cells to total aliphatic acid can be substantially increased; and thus, the gene can be applied to production of high-quality biodiesel (with low burning point and low viscosity and other related products.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a Chlamydomonas reinhardtii hemolytic phosphatidic acid acyltransferase, and also relates to the use of the enzyme. Background technique [0002] About 80% of the world's energy today comes from fossil fuels. Since the industrial revolution in the 19th century, fossil fuels have been gradually exhausted. As a non-renewable resource, human beings must find a way to replace fossil fuels and become The main energy source will not change the industrial structure and the replacement of productivity tools too drastically. Among the many alternative energy sources, biofuels have received the most attention, including biodiesel. [0003] At present, the biomass that can be used to make biodiesel is mainly higher plants, such as rapeseed, soybean, palm oil, sunflower, jatropha and so on. Although these plants have high oil production capacity, they need a large area o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N1/13C12N1/19C12P7/64
CPCC12N9/1029C12P7/649Y02E50/10
Inventor 尹康燮李中泽韩丹翔胡强
Owner GUOTOU BIO TECH INVESTMENT CO LTD
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