Method for synthesizing acrylic acid from cyanobacteria

A technology of acrylic acid and cyanobacteria, applied in the field of synthetic biology, can solve the problems of inability to realize large-scale production, environmental pollution, unsustainability, etc., and achieve the effects of easy industrial transformation, good purity, and easy production cost

Inactive Publication Date: 2018-11-02
嘉兴欣贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this method has many advantages, the raw material depends on traditional fossil energy, which causes heavy pollution, high energy consumption and is unsustainable.
At present, there are also semi-biosynthetic methods or total biosynthesis methods to s

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  • Method for synthesizing acrylic acid from cyanobacteria
  • Method for synthesizing acrylic acid from cyanobacteria
  • Method for synthesizing acrylic acid from cyanobacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Plasmid construction process: According to the NSI gene sequence of cyanobacterium Syn7942, according to the attached figure 2 The plasmid map in the construction of the integration vector NSI. Then, according to the gene ceaS2 amino acid sequence provided in the present invention, design the nucleic acid sequence of the corresponding gene according to the codon preference of the cyanobacterium Syn7942 cell, send the gene ceaS2 to a commercial company for synthesis, design a homology arm of about 20 bp size, and follow the attached image 3 In the plasmid map, the two genes were constructed on the integration vector NSI using a one-step cloning kit.

Embodiment 2

[0065] Embodiment 2 conversion process:

[0066] 1) Take 2 mL of wild-type Syn7942 with a growth density OD730 of 0.8-1.2 in a centrifuge tube, then centrifuge at 10,000 rpm for 2 min, and remove the supernatant;

[0067] 2) Mix the precipitate in the previous step with 1mL 10mM sodium chloride solution, then centrifuge at 10000rpm for 2min, and remove the supernatant;

[0068] 3) Mix the precipitate in the previous step with 1 mL of sterilized anti-BG11-free liquid medium, then centrifuge at 10,000 rpm for 2 min, and remove the supernatant;

[0069] 4) Mix the precipitate in the previous step with 100 uL of sterilized anti-BG11-free liquid medium, and then add 200 ng of plasmid DNA to it. Thoroughly seal the centrifuge tube with tinfoil (protected from light), then incubate in a shaker at 100 rpm at 30°C for 10 hours;

[0070] 5) Apply all the cyanobacteria in the centrifuge tube in the previous step to the BG11 solid medium containing 25ug / mL chloramphenicol, put the plate...

Embodiment 3

[0072] The verification of embodiment 3 transgenic cyanobacteria and the detection process of product:

[0073] Pick about 10 monoclonal cyanobacterial plaques grown on BG11 solid medium containing 25ug / mL chloramphenicol, insert them into 5mL25ug / mL chloramphenicol BGS1 liquid medium for culture, and extract the genome after the cyanobacteria are cultivated , verify the ceaS2 target gene by PCR with the designed primers, and send the product to sequence by PCR to determine whether the gene sequence is correct; insert the verified correct ceaS2-transformed cyanobacterium Syn7942 into 100mL BG11 liquid medium for light culture, and wait for the OD730 to be 1 , add the substrate p-hydroxyphenylpropionic acid, and add IPTG to induce the expression of two ceaS2 genes. The expressed enzyme uses glyceraldehyde triphosphate or dihydroxyacetone phosphate produced by photosynthesis of cyanobacteria as the substrate to synthesize acrylic acid, and then respectively takes After the induc...

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Abstract

The invention relates to a method for synthesizing acrylic acid from cyanobacteria. The method comprises the following steps: 1), firstly, based on an NSI gene of cyanobacteria Syn7942, constructing an integrated vector NSI, and recombining to obtain a vector NSI-ceaS2; 2), transforming the recombinant vector NSI-ceaS2 into cyanobacteria Syn7942 cells, and then preliminarily screening out monoclonal transgenic cyanobacteria Syn7942 through a chloramphenicol solid culture medium; 3), transferring the screened monoclonal transgenic cyanobacteria Syn7942 to a liquid culture medium with chloramphenicol resistance, and after the cyanobacteria grow out, extracting a cyanobacteria genome for PCR verification of a target gene; 4), transferring the successfully verified monoclonal transgenic cyanobacteria Syn7942 to the liquid culture medium for culture, and when the cyanobacteria grow till OD730 is greater than or equal to 1, adding IPTG for inducing expression of a gene ceaS2, and catalytically synthesizing the acrylic acid by an enzyme by using glyceraldehyde-3-phosphate (G3P) or dihydroxyacetone phosphate (DHAP) produced by photosynthesis of the cyanobacteria as a substrate; 5), performing separation and purification. By the method, when the acrylic acid is synthesized through transformation of the cyanobacteria as substrate organisms, only sunlight and moisture and the like are required as production raw materials, production equipment and a production environment are easy to construct, and high electricity consumption is not required, so that the production cost of the methodprovided by the invention is greatly lower than those of other production methods.

Description

technical field [0001] The invention relates to a method for synthesizing acrylic acid by using cyanobacteria, belonging to the field of synthetic biology. Background technique [0002] Cyanobacteria (Cyanobacteria) is a type of bacteria that obtain energy through photosynthesis, also known as blue-green algae, blue-green bacteria or cyanobacteria. Chloroplastin makes it appear blue-green. Cyanobacteria are the earliest algae that appeared on the earth. They are the simplest and most primitive single-celled organisms. Simple photosynthesis unit. [0003] Due to the unique physiological characteristics of cyanobacteria capable of photosynthesis, many scientists regard cyanobacteria as a "biological photoreactor", through which carbon dioxide and water in the air can be converted into chemical products and some high-value natural products needed by humans. . Genetic modification of cyanobacteria to biosynthesize these products has many advantages: (1) At present, the genom...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12P7/40C12R1/01
CPCC12N15/74C12P7/40
Inventor 蒿飞朱文博陈贤情夏文豪杨月王筱王文杨慧江会峰
Owner 嘉兴欣贝莱生物科技有限公司
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