Method for producing caffeic acid by using blue algae

A technology of caffeic acid and cyanobacteria, applied in the field of genetic engineering

Inactive Publication Date: 2015-06-17
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the uncertainty of expressing the target gene in cyanobacteria, there are currently no reports on the use of cyanobacteria to produce caffeic acid

Method used

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  • Method for producing caffeic acid by using blue algae
  • Method for producing caffeic acid by using blue algae
  • Method for producing caffeic acid by using blue algae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The sref8 gene synthesis of embodiment 1 codon optimization

[0052] According to the codon preference of Synechocystis sp., codon optimization was performed on the ref8 gene of Arabidopsis thaliana, and the sref8 gene was synthesized. The nucleotide sequence is shown in SEQ ID NO.1. The amino acid sequence of the C3H protein expressed by the sref8 gene is shown in SEQ ID NO.2. For codon optimization, see figure 1 , 23% of the coding sequence in the ref8 gene was optimized. During the optimized synthesis, an EcoRI restriction site was added to the 5' end of the sref8 gene, and a PstI restriction site was added to the 3' end.

Embodiment 2

[0054] Isolate and clone Synechocystis double homologous recombination upstream and downstream arm gene fragments, psbA2 promoter and Escherichia coli terminator T1T2 cDNA fragments used to construct the expression vector of transgenic Synechocystis sp. PCC6803. The DNA polymerase used in the PCR amplification fragment in this experiment was Phusion High-Fidelity DNA Polymerase (purchased from NEB Company).

[0055] Cloning of upstream and downstream arm gene fragments of Synechocystis sp. PCC6803 homologous recombination:

[0056] The homologous recombination arms are the upstream and downstream sequences near slr1285 of the Synechocystis sp. PCC6803 genome, and the amplified fragment sizes are 594bp and 606bp respectively (sequences such as SEQ ID NO: 13, and SEQ ID NO: 14, Synchocystis sp.PCC6803) amplification primers are designed as follows:

[0057] Upper arm forward primer: 5'-ATCggtaccGGCAATGCAATTAATTAAAAATGGC-3' (contains KpnI restriction site), (SEQ ID NO.3)

[0...

Embodiment 3

[0075] Example 3 Homologous Recombination Vector Construction

[0076] The 5ST1T2 positive clone (containing the T1T2 fragment) obtained in Example 2 was double-digested with pstI and BamHI to recover the T1T2 fragment; the pBluescript II SK (+) plasmid was also treated with pstI and BamHI restriction endonucleases to recover the vector fragment 1. The recovered T1T2 fragment was ligated with vector fragment 1 to obtain pBluescript SK T1T2 plasmid.

[0077] The homologous recombination upstream arm-positive clone obtained in Implementation 2 was double digested with KpnI and XhoI, and the upstream arm fragment was recovered. The pBluescript SK T1T2 plasmid was also double digested with KpnI and XhoI, and the vector fragment 2 was recovered. The recovered upstream arm fragment was ligated with vector fragment 2 to obtain pBluescript SK T1T2-ups plasmid.

[0078] The homologous recombination downstream arm-positive clone obtained in Implementation 2 was double digested with S...

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Abstract

The invention relates to a method for producing caffeic acid by using blue algae, which comprises the following steps of 1) inserting relevant gene segments in plasmid pBluescript II SK+ to obtain recombined blank plasmids; 2) inserting gene segments of nucleotide sequences such as SEQ ID NO.1 among enzyme cutting sites of the EcoRI-PstI incision enzymes of the recombined blank plasmids obtained in the step 1 to obtain recombined plasmids; 3) transforming the recombined plasmids into Synechocystis sp. PCC6803 to obtain transgenic synechocystis; and 4) conducting enlarging cultivation on the transgenic synechocystis obtained in the step 3 and then conducting extraction to obtain the caffeic acid. By constructing homologous recombination carriers for Synechocystis sp. PCC6803 transformation, algal strains capable of producing the caffeic acid under the condition that precursor substance p-coumaric acid is added are obtained through synechocystis transformation, and a new means is provided for the production of the caffeic acid.

Description

technical field [0001] The invention relates to a method for producing caffeic acid by using cyanobacteria, in particular to a method for producing caffeic acid by using Synechocystis sp. PCC6803, which belongs to the technical field of genetic engineering. Background technique [0002] Caffeic acid is a phenylpropane secondary metabolite produced by plants under external stimuli (such as pathogenic bacteria infection, plant wounds, ultraviolet radiation, strong light stress, nutritional deficiencies, and low temperatures). Caffeic acid and its derivatives caffeic acid phenethyl ester have strong antioxidant, anticancer, anti-inflammatory and immunomodulatory activities (Gulcin, 2006; Rajendra et al., 2011; Chao et al., 2009; Park et al. , 2004), is a kind of pharmaceutical intermediates and raw materials that have a wide range of effects in medicine. [0003] Caffeic acid has antibacterial and antiviral effects, and also has a wide range of antibacterial effects, but it ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/42C12N15/74C12R1/01
Inventor 何庆芳张燕薛勇毕玉平陈高葛海涛杨连群范仲学王俊燕于金慧董学卫彭振英边斐
Owner 山东省农业科学院高新技术研究中心
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