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A kind of recombinant cyanobacteria expressing fatty acid with high efficiency and preparation method thereof

A technology of cyanobacteria fatty acid and cyanobacteria, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of synthetic fatty acid tolerance crossover, inability to achieve industrial production, and low efficiency of fatty acid, so as to improve Tolerance, convenience, economic stability, effect of reducing ROS content

Active Publication Date: 2021-08-17
SHANGHAI JIAOTONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the efficiency of fatty acid production by cyanobacteria is low at the present stage, and at the same time, the tolerance of engineered cyanobacteria to self-synthesized fatty acids is crossed, which cannot meet the needs of industrial production

Method used

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  • A kind of recombinant cyanobacteria expressing fatty acid with high efficiency and preparation method thereof
  • A kind of recombinant cyanobacteria expressing fatty acid with high efficiency and preparation method thereof
  • A kind of recombinant cyanobacteria expressing fatty acid with high efficiency and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Constructing a Genome Integration Plasmid Platform

[0048] Synechocytis sp. PCC 6803 genomic DNA was extracted and used as a template to clone Synechocytis sp. related genes by PCR, including recombinant homology arms U0168, D0168, PcpcB, ssSec, Lgt (GenbankAccession No.CP003265.1). The AcTesA gene sequence was synthesized by Jinweizhi Company. The DNA resistance screening marker nptII fragment was derived from the PCR product of the existing vector plasmid pRSFDuet-1Vector (purchased from Novagen). Through enzyme digestion, the desired DNA fragments of each gene with relevant cohesive ends were obtained. The primer sequences used above are as follows:

[0049]

[0050]

[0051] Through vector construction, U0168, PcpcB, ssSec, Lgt, and AcTesA sequences were sequentially cloned into the multiple cloning sites KpnI and NotI of the plasmid pBluescript II KS (+) by enzyme digestion and ligation to form a plasmid with PcpcBssSecLgtAcTesA, and nptII Ligat...

Embodiment 2

[0052] The acquisition of embodiment 2 transformant mAcT

[0053] Through homologous recombination, the PcpcBssSecLgtAcTesA-nptII sequence was integrated into Synechocytis sp. PCC6803 genomic DNA and expressed (each chromosome contains at least one copy of PcpcBssSecLgtAcTesA-nptII). Specific steps are as follows:

[0054] 1) Transformation: collect Synechocytis sp.PCC 6803 cyanobacteria cells in the logarithmic growth phase, and adjust the concentration of algae liquid to OD with BG11 liquid medium 730 =2.5, take 5-20 μg of plasmid PcpcBssSecLgtAcTesA-nptII and add it to 500 μl of algae liquid and mix well, 25 μmol / m 2 Under the light intensity of / s, it was cultured statically for 2 hours and shaken on a shaker overnight.

[0055] 2) Screening: recombinants were screened on a BG11 solid medium plate containing kanamycin, and the screening concentration of kanamycin was 50ug / ml. Homogenized transformants were selected after multiple passages, and the screened recombinant w...

Embodiment 3

[0058] Example 3 total membrane protein extraction and separation of inner and outer membrane proteins:

[0059] 1. After the cyanobacteria were cultured for 24 hours, the cells were collected and centrifuged at 6700g for 10 minutes at 4°C;

[0060] 2. Wash the cells twice with 100mM Tris-HCl (pH7.5) equivalent to 50 times the volume of the cells;

[0061] 3. Resuspend the cells with 0.5-1ml of 10mM Tris (pH7.5), and freeze the cell resuspension at -80°C for 2 hours;

[0062] 4. Sonicate the cells (period: 15s sonication, 45s cooling), until the cell suspension changes from turbid to translucent, centrifuge at 10000g centrifugal force for 10 minutes at 4°C, and collect the supernatant;

[0063] Centrifuge at 5.4°C, 100,000g centrifugal force for 10 minutes, the supernatant is the cytoplasmic protein, and the lower layer is the total membrane protein;

[0064] 6. Wash the total membrane protein twice with 500 μl of 10mM Tris (pH7.5), resuspend with 100mM Tris-HCl (pH7.5), and...

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Abstract

The invention discloses a recombinant cyanobacterium with high-efficiency expression of fatty acid and a preparation method thereof, and relates to the field of fatty acid production. A recombinant cyanobacteria is obtained by constructing a plasmid for increasing the expression level of fatty acid in cyanobacteria, and the recombinant cyanobacteria overexpresses prolipoprotein diacyltransferase Lgt and thioesterase AcTesA. Prolipoprotein diacyltransferase Lgt is a scaffold protein that localizes to the cell membrane under the action of a cyanobacterial membrane targeting sequence. The thioesterase AcTesA is connected to the scaffold protein Lgt through the flexible peptide FL3. The thioesterase protein AcTesA has low substrate specificity, can hydrolyze the C8-C16 acyl ACP, and can obtain a variety of fatty acid products. The invention fixes the thioesterase protein AcTesA on the cell membrane through the scaffold protein Lgt, accelerates the fatty acid synthesis rate in the cyanobacteria cell, reduces the ROS content in the cell, and finally increases the fatty acid output secreted by the transgenic mutant strain outside the cell.

Description

technical field [0001] The invention relates to a recombinant cyanobacterium, in particular to its effect on improving the synthesis efficiency of cyanobacteria fatty acid and its preparation method. Background technique [0002] Energy is the driving force for the development of the entire human society. With the shortage of petroleum resources and the deterioration of the environment, the demand for environmentally friendly and renewable energy is becoming more and more urgent, and biofuels can solve this problem well. Fatty acids are important precursors for the synthesis of biofuels, which can be further processed into chemical materials and energy sources for different purposes such as fatty alcohols, fatty hydrocarbons, and fatty acid methyl esters through chemical or biosynthesis. So far, the microorganisms used to produce fatty acids are mainly Escherichia coli and yeast, but natural strains cannot efficiently synthesize fatty acids, and the oil production is limited...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21C12P7/40C12P7/64C12N9/16C12N9/10C12R1/01
CPCC12N9/1029C12N9/16C12N15/74C12P7/40C12P7/6418C12Y301/02
Inventor 马钢莎加·阿夫林王毓舒贺林
Owner SHANGHAI JIAOTONG UNIV
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