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Method for producing three-carbon compounds by using gene engineering cyanobacteria

A three-carbon compound, genetic engineering technology, applied in genetic engineering, biochemical equipment and methods, botanical equipment and methods, etc., can solve problems such as time-consuming and cost, achieve cost saving of raw materials, avoid consumption, and broad application prospects. Effect

Inactive Publication Date: 2015-06-24
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the collection, treatment, and decomposition of biomass resources are time-consuming and costly
However, there is no direct conversion of CO using genetically engineered cyanobacteria. 2 Reports on the production of important three-carbon compounds such as dihydroxyacetone, 3-hydroxypropionic acid, 1,3-propanediol, d-glycerate, and hydroxypyruvate

Method used

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  • Method for producing three-carbon compounds by using gene engineering cyanobacteria
  • Method for producing three-carbon compounds by using gene engineering cyanobacteria
  • Method for producing three-carbon compounds by using gene engineering cyanobacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of plasmid pAM-YW2 carrying genes related to the synthesis pathway of dihydroxyacetone

[0051] 1) Extract pAM2991 plasmid

[0052] Escherichia coli DH5α (pAM2991) carrying the pAM2991 plasmid was inoculated into 5 mL of LB liquid medium containing spectinomycin (100 μg / mL) according to the inoculum size of 1%, and cultured in a shaker at 200 rpm at 37 ° C for 12 h . The pAM2991 plasmid was extracted from the cultured cells.

[0053] 2) Amplify the Ptrc promoter

[0054] Using synthetic primers, the Ptrc promoter was amplified by PCR from the pAM2991 plasmid.

[0055] Ptrc upstream primer 5'-AGATCTGACAGCTTATCATCGACT-3', carrying a BglII restriction site;

[0056] The downstream primer of Ptrc, 5'-CTCGAGTTCCATGGTCTGTTTCCT-3', carries an XhoI restriction site.

[0057] The above-mentioned Ptrc promoter has a sequence length of 268 bases, and its nucleotide sequence is shown in SEQ ID NO.9.

[0058] 3) Construction of pAM-P2 plasmid

[0059] ...

Embodiment 2

[0072] Example 2: Construction of plasmid pAM-YW3 carrying genes related to 3-hydroxypropionic acid synthesis pathway

[0073] 1) extract pAM-P2-gpp1 plasmid,

[0074] Escherichia coli DH5α (pAM2991) carrying the pAM-P2-gpp1 plasmid was inoculated into 5 mL of LB liquid medium containing spectinomycin (100 μg / mL) according to the inoculum size of 1%, and shaken at 200 rpm at 37°C. Cultured in bed for 12h. The pAM-P2-gpp1 plasmid was extracted from the cultured cells.

[0075] 2) Amplification of glycerol dehydratase gene dhaB

[0076] Genomic DNA of Clostridium butyricum DSM 10702 was prepared by a conventional method, and glycerol dehydratase gene dhaB was obtained by PCR amplification from the genomic DNA of Clostridium butyricum DSM 10702 using synthetic primers.

[0077] dhaB upstream primer 5'-CTCGAGATGATAAGTAAAGGATTTAG-3', carrying an XhoI restriction site;

[0078]The dhaB downstream primer 5'-GCGGCCGCTTACTCAGCTCCAATTGTGC-3' carries a NotI restriction site.

[0079...

Embodiment 3

[0087] Embodiment 3: the transformation of cyanobacteria and the screening of genetically engineered cyanobacteria

[0088] 1) Transformation of cyanobacteria strains

[0089] Synechococcus elongatus PCC 7942 was grown to OD 730nm 0.4-0.8, take 10mL of cyanobacteria culture solution, centrifuge at 5,000×g for 5 minutes, remove the supernatant to collect the bacteria; wash once with sterile 10mM NaCl, resuspend the bacteria with 5mL of fresh BG11 medium; take 500μL of cyanobacteria to resuspend Put the suspension into a 1.5mL centrifuge tube, add the recombinant plasmid with a final concentration of 100ng / mL, mix well, and incubate in the dark at 30°C for 12-18 hours; spread the mixture of cyanobacteria suspension and recombinant plasmid DNA on the BG11 solid Culture medium (containing spectinomycin 20 μg / mL), 30°C, 100 μE·s -1 m -2 Cultivate for 10-14 days under a certain light intensity until a single colony appears on the BG11 solid medium.

[0090] 2) Screening of genet...

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Abstract

The invention discloses a method for producing three-carbon compounds by directly using gene engineering cyanobacteria to transform CO2. Thee method is characterized in that related genes in a synthesis approach of the three-carbon compounds are transformed to cyanobacteria, and gene engineering cyanobacteria is prepared, and subjected to enlarged culture for producing the three-carbon compounds such as dihydroxy acetone, 3-hydracrylic acid, 1,3-propylene glycol, d-glyceric acid and hydroxypyruvic acid. The method avoids high-cost collection, treatment and decomposition processes of a biomass resource, directly uses the low-cost raw material CO2 as a carbon source, produces the widely applied chemicals and fuels, can reduce the emission of CO2 in atmosphere, and has popularization and application values.

Description

technical field [0001] The invention relates to a method for producing three-carbon compounds by using genetically engineered cyanobacteria, in particular to a method for directly converting CO by using genetically engineered cyanobacteria 2 Process for producing three carbon compounds. Background technique [0002] Three-carbon compounds, such as dihydroxyacetone, 3-hydroxypropionic acid, 1,3-propanediol, d-glyceric acid and hydroxypyruvic acid, etc., have a wide range of uses in chemical, material, pharmaceutical and textile industries, and can be used as chemical Feedstocks for synthesis and biocatalysis are used to produce high-value products such as various chemicals and fuels. Dihydroxyacetone is widely used in the cosmetic industry, and it is also widely used in the synthesis of chemicals and biomaterials; 3-hydroxypropionic acid is one of the 12 most promising platform compounds screened by the U.S. Department of Energy, which can be used for synthesis Acrylic acid...

Claims

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Application Information

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IPC IPC(8): C12P7/28C12P7/42C12P7/18C12N15/74C12N15/60C12N15/55C12N15/53
CPCC12P7/18C12P7/28C12P7/42Y02P20/141
Inventor 许平王钰陶飞唐鸿志
Owner SHANGHAI JIAO TONG UNIV
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