Method using blue algae to synthesize phloretin

A phloretin and cyanobacteria technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of inability to achieve large-scale production, low phloretin efficiency, and chemical waste liquid discharge, etc. The effect of industrial transformation, simple operation and production cost reduction

Pending Publication Date: 2019-06-21
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

The synthesis of phloretin by these chemical methods has problems such as low efficiency, discharge of chemical waste liquid, and serious environmental pollution.
There are also some methods of biosynthesizing phloretin by fermentation of Escherichia coli or Saccharomyces cerevisiae. Although these biosynthesis methods solve the problems of low yield and some environmental pollution, due to the cost of raw materials, equipment, water and electricity in the production process Too high for mass production

Method used

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  • Method using blue algae to synthesize phloretin
  • Method using blue algae to synthesize phloretin
  • Method using blue algae to synthesize phloretin

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Embodiment 1

[0035] This embodiment provides the vector plasmid construction process: according to the method provided in the present invention Synechococcus elongatus The NSI gene sequence of PCC7942 (Syn7942 for short), according to the attached figure 2 The plasmid map in the construction of the integration vector NSI, and then according to the amino acid sequence of 4CL and CHS provided in the present invention, the nucleic acid sequence of the corresponding gene is designed according to the codon preference of Syn7942 cells, and the gene 4CL and CHS are sent to companies with gene synthesis capabilities on the market Synthesize, design a homology arm with a size of about 20bp, 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.

[0036] NSI as Synechococcus provided by the present invention Synechococcus elongatus A gene sequence on the PCC7942 genome, the role of NSI is that changes in this gen...

Embodiment 2

[0039] This embodiment provides the recombinant transformation process:

[0040] 1) Take 2 mL of wild-type Syn7942 with a growth density of OD730 of 0.8-1.2 in a centrifuge tube, then centrifuge at 10,000 rpm for 2 min, and remove the supernatant;

[0041] 2) Mix the precipitate in the previous step with 1mL 10mM sodium chloride solution, then centrifuge at 10000rpm for 2min, and remove the supernatant;

[0042] 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;

[0043] 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. Seal the centrifuge tube thoroughly with tinfoil (protect from light), and then incubate in a shaker at 100 rpm at 30°C for 10 hours;

[0044] 5) Apply all the cyanobacteria in the centrifuge tube in the previous step to the BG11 solid medium containing 2...

Embodiment 3

[0047] This embodiment provides the screening confirmation of transgenic cyanobacteria and the detection process of the product:

[0048] Pick about 10 monoclonal cyanobacterial plaques grown on BG11 solid medium containing 25ug / mL chloramphenicol, insert them into 5mL 25ug / mL chloramphenicol BG11 liquid medium for culture, and extract For the genome, verify the two target genes of CHS and 4CL by PCR with the designed primers, and send the products to sequence by PCR to determine whether the gene sequence is correct; insert the Syn7942 cyanobacteria that have been verified to have the correct transgenic CHS gene and 4CL gene into 100mL BG11 liquid medium Medium light culture, when the OD730 was 1, the substrate p-hydroxyphenylpropionic acid was added, and IPTG was added to induce the expression of CHS and 4CL genes, and then the transgenic cyanobacteria were cultured for 1 day, 3 days, and 5 days after induction, respectively, and used The high-pressure crusher breaks the cyan...

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Abstract

The invention provides a technical method using 'photosynthetic bacteria', namely blue algae as the substrate organism to synthesize phloretin. The method mainly includes: exogenously expressing 4 coumarate Coenzyme A Ligase (4CL) and Chalcone synthase (CHS) which are two key enzymes for phloretin synthesis in the blue algae so as to use p-hydroxybenzene propanoic acid (also called phloretic acid)as the substrate to catalytically synthesize the phloretin, the 4CL catalyzes single-molecule p-hydroxybenzene propanoic acid to generate p-hydroxybenzene propionyl coenzyme A, the CHS catalyzes three-molecule malonyl coA and the single-molecule p-hydroxybenzene propionyl coenzyme A to synthesize the single-molecule phloretin. The method has the advantages that the phloretin is produced by usingone microorganism which can perform photosynthesis, the phloretin biological synthesizing approach which is cheap in raw material, simple in equipment, low in environment pollution and high in yield is created, and the method conforms to the direction of green production.

Description

technical field [0001] The invention belongs to the technical field of phloretin synthesis, and in particular relates to a method for producing phloretin by using blue algae. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/26C12N1/21C12R1/01
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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