Method for synthesizing tagatose from cyanobacteria

A technology of tagatose and cyanobacteria, applied in the field of synthetic biology, can solve the problems of difficult industrialized large-scale production, many by-products under alkaline conditions, and difficult product separation, and achieve easy industrial transformation, good purity, scale and The effect of a small amount

Pending 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

AI Technical Summary

Problems solved by technology

[0005] At present, the synthesis methods of tagatose are mainly chemical catalysis synthesis method and biotransformation method. The chemical catalysis synthesis method uses galactose as raw material, through metal hydroxide (usually Ca (OH) CaCl2) catalyzes galactose to generate tagatose-Ca complexes, and then releases tagatose from the complexes through acid neutralization. This method has the advantages of expensive raw materials, chemical pollutants, and many by-products under alkaline conditions. Difficult to separate and many other disadvantages
The biotransformation method uses L-arabinose isomerase to convert galactose into tagatose, but the same reaction has the disadvantages of expensive raw materials, low conversion efficiency, and difficult product separation, making it difficult to realize large-scale industrial production.

Method used

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  • Method for synthesizing tagatose from cyanobacteria
  • Method for synthesizing tagatose from cyanobacteria
  • Method for synthesizing tagatose from cyanobacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 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 amino acid sequences of the genes HXK, FbaA and phytase provided in the present invention, the nucleic acid sequences of the corresponding genes are designed according to the codon preference of Cyanobacteria Syn7942 cells, and the genes HXK, FbaA and phytase are sent to commercial companies for synthesis, and the same size of about 20bp is designed. source arm, follow the attached image 3 In the plasmid map, the three genes were constructed on the integration vector NSI using a one-step cloning kit.

Embodiment 2

[0054] Conversion process:

[0055] 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;

[0056] 2) Mix the precipitate in the previous step with 1mL 10mM sodium chloride solution, then centrifuge at 10000rpm for 2min, and remove the supernatant;

[0057] 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;

[0058] 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;

[0059] 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 in a light i...

Embodiment 3

[0062] Verification of transgenic cyanobacteria and product detection process:

[0063] Pick about 10 monoclonal cyanobacterial plaques grown on BG11 solid medium containing 25ug / mL chloramphenicol, insert them into 5mL25ug / mL chloramphenicol BG11 liquid medium for culture, and wait until the cyanobacteria are cultivated until the OD730 is 0.5 , extract the genome, verify the three target genes of HXK, FbaA and phytase by PCR with the designed primers, and send the products to sequence by PCR to determine whether the gene sequence is correct; transfer the correct verified monoclonal transgenic cyanobacterium Syn7942 into 100mL BG11 Culture in liquid medium under light. When OD730 is S, add IPTG to induce the expression of three genes, HXK, FbaA and phytase. The expressed enzymes will synthesize tagatose with fructose produced by photosynthesis of cyanobacteria, and then take the induced The transgenic cyanobacteria are cultured for 1 day, 3 days, and 5 days, and the cyanobacte...

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Abstract

The invention relates to a method for synthesizing tagatose from cyanobacteria. The method comprises the following steps: 1), firstly, according to an NSI gene of cyanobacteria Syn7942, constructing an integrated vector NSI, and then inserting HXK, FbaA and phytase genes into the vector NSI to obtain a required recombinant vector NSI-HFP; 2), transforming the vector NSI-HFP 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 extracting a cyanobacteria genome for PCR verification of a target gene; 4), culturing the monoclonal transgenic cyanobacteria Syn7942 under a proper condition, and adding IPTG for inducing expression of the HXK, FbaA and phytase genes, and synthesizing the tagatose by enzymes by using fructose produced by photosynthesisof the cyanobacteria as a substrate; 5), performing separation and purification. By the method, when the tagatose 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 the production cost is greatly reduced.

Description

technical field [0001] The invention relates to a synthesis technology of tagatose, in particular to a method for producing tagatose 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12P19/02C12R1/01
CPCC12N15/74C12P19/02
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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