Construct, vector and cyanobacteria for synthesizing caffeic acid, and method for producing caffeic acid in cyanobacteria

A technology of caffeic acid and cyanobacteria, applied in the field of caffeic acid production, can solve problems such as high cost and inability to carry out large-scale production, and achieve the effects of reduced production cost, simple construction of production equipment and production environment, and simple product purification process

Pending Publication Date: 2022-03-04
NINGBO WOMEN & CHILDREN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it needs the support of external carbon sources for coumaric acid, and the cost is high, so it cannot be mass-produced

Method used

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  • Construct, vector and cyanobacteria for synthesizing caffeic acid, and method for producing caffeic acid in cyanobacteria
  • Construct, vector and cyanobacteria for synthesizing caffeic acid, and method for producing caffeic acid in cyanobacteria
  • Construct, vector and cyanobacteria for synthesizing caffeic acid, and method for producing caffeic acid in cyanobacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This embodiment provides the construction process of the vector plasmid: the upstream and downstream of slr2081, Ome and PrbcL gene sequences of Synechocystissp. SEQ ID NO.2, see SEQ ID NO.3 for the spectinomycin gene sequence, see SEQ ID NO.4 for the strong promoter PrbcL sequence, insert the spectinomycin resistance gene and the strong promoter sequence PrbcL into the upstream gene sequence of slr2081 and Between the downstream gene sequences of slr2081, the basic structure of the vector is as follows figure 2 shown. according to figure 2 Plasmid map in Synechocystis sp. PCC6803 gene expression platform pJS410 was constructed.

[0038]According to the PAL, C4H and REF8 gene sequences, the nucleic acid sequences of the corresponding genes were designed according to the codon preference of Synechocystis sp. PCC6803 cells. The phenylalaninase gene sequence (PAL) is shown in SEQ ID NO.5, the cinnamic acid-4-hydroxylase gene sequence (C4H) is shown in SEQ ID NO.6, and...

Embodiment 2

[0042] This embodiment provides the recombinant transformation process:

[0043] 1) The wild-type Synechocystis sp. PCC6803 is cultivated in a constant temperature and light incubator until it grows vigorously (OD730=0.6-0.8 or OD600=1.1-1.2). Take out 30mL Syn6803 algae liquid, centrifuge at 4500rpm for 15min, and remove the supernatant;

[0044] 2) Wash the algae twice with an equal volume of fresh BG11 medium without antibiotics, centrifuge at 4500rpm for 15min, and remove the supernatant;

[0045] 3) Add 1mL BG11 to resuspend the centrifuged algae by pipetting and transfer to a 1.5mL sterilized EP tube, carefully add about 10μg of plasmid pJS410-PAL-C4H-REF8 into it, shake and mix;

[0046] 4) Cultivate in light for at least 4 hours in a light incubator;

[0047] 5) After about 4 hours, spread a pre-sterilized 0.45 μm cellulose filter membrane on the BG11 solid plate, and discharge the air bubbles between the filter membrane and the solid medium. Divide 1mL of the cyano...

Embodiment 3

[0053] This example provides the screening confirmation of transgenic cyanobacteria and the detection process of the product: pick about 10 monoclonal cyanobacterial plaques grown on BG11 solid medium containing 10 μg / mL spectinomycin, and insert 5 mL of 20 μg / mL spectinomycin Cultivate in BG11 liquid medium. After the cyanobacteria are cultured, extract the genome and use the designed primers to verify the three target genes of PAL, C4H and REF8 by PCR, and send the products to sequence by PCR to determine whether the gene sequence is correct; the correct one will be verified The transgenic cyanobacteria Syn6803 with PAL, C4H and REF8 genes were inserted into 100mL BG11 liquid medium for light culture. When the OD730 was 1, the cyanobacteria cells were broken with a cell disruptor, separated and purified, and detected with a high performance liquid chromatography.

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Abstract

The invention provides a construction body for synthesizing caffeic acid, the construction body comprises a promoter with activity in cyanobacteria, and a phenylalanase gene, a cinnamic acid-4-hydroxylase gene and a p-coumaric acid-3-hydroxylase gene which are controlled by the promoter, the sequence of the phenylalanase gene is shown as SEQ ID NO.5, the sequence of the cinnamic acid-4-hydroxylase gene is shown as SEQ ID NO.5, and the sequence of the p-coumaric acid-3-hydroxylase gene is shown as SEQ ID NO.5. The gene sequence of the cinnamic acid-4-hydroxylase is as shown in SEQ ID NO.6, and the gene sequence of the p-coumaric acid-3-hydroxylase is as shown in SEQ ID NO.7. The invention further discloses a preparation method of the cinnamic acid-4-hydroxylase. A carrier and cyanobacteria and a method of producing caffeic acid in cyanobacteria. The invention also relates to a vector comprising the construct, cyanobacteria comprising the construct or transformed with the vector, and a method for producing caffeic acid in cyanobacteria. According to the construction body for synthesizing the caffeic acid, a chassis organism applying the construction body can catalyze CO2 to directly synthesize the caffeic acid, an external carbon source does not need to support cumaric acid, the cost is low, and the construction body is suitable for large-scale production.

Description

technical field [0001] The invention relates to the technical field of caffeic acid synthesis, in particular, to a construct, carrier, cyanobacteria and a method for producing caffeic acid in the cyanobacteria for synthesizing caffeic acid. Background technique [0002] Cyanobacteria, as a prokaryotic organism, can perform photosynthesis like higher plants. Many scientists regard cyanobacteria as a "photosynthetic reactor". Cyanobacteria only fix CO 2 It can biosynthesize a large number of chemical products and natural plant products required by humans. Genetic modification of cyanobacteria for biosynthesis has many advantages: (1) The genomes of common cyanobacteria such as Anabaena and Synechocystis have been sequenced. Due to the simple structure of cyanobacteria, scientists have developed a set of relevant The gene editing system of blue-green algae; (2) blue-green algae grows fast and is suitable for large-scale production; (3) blue-green algae have strong adaptability...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/67C12N15/60C12N15/53C12N1/21C12P7/42C12R1/01
CPCC12N15/74C12N15/65C12N15/52C12N9/0073C12N9/88C12N9/0071C12Y403/01024C12Y114/13011C12Y114/17C12P7/42C12N2800/22C12N2830/008
Inventor 邱海燕吴军华高恶斌
Owner NINGBO WOMEN & CHILDREN HOSPITAL
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