Engineering bacteria producing l-rhamnose and its construction method and application

A technology of rhamnose isomerase and rhamnose, which is applied in the field of genetic engineering, can solve the problems of many by-products, high cost, and affecting the yield and output of rhamnose, and achieve the effect of avoiding hydrolysis steps and reducing the burden

Active Publication Date: 2021-11-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, there are certain problems in these three methods, which affect the yield and yield of rhamnose
Although rhamnose has extremely important economic value, the current extraction process of rhamnose is complex, with many by-products, high cost and low yield, and there is still no suitable method for large-scale industrial production of rhamnose

Method used

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  • Engineering bacteria producing l-rhamnose and its construction method and application
  • Engineering bacteria producing l-rhamnose and its construction method and application
  • Engineering bacteria producing l-rhamnose and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1. Construction of recombinant plasmids co-expressing rhamnose-1-phosphate aldolase, fructose-1-phosphatase and L-rhamnose isomerase

[0099] 1. Using the Escherichia coli K12 genome as a template, the primer pair P1 and P2 are used for PCR amplification, and the gene encoding fructose-1-phosphatase (yqaB) is obtained by PCR amplification. The PCR conditions were as follows: 98°C, 2min; 98°C, 20sec, 55°C, 20sec, 72°C, 30sec (30 cycles); 72°C, 5min. Detected by 1% agarose gel electrophoresis, the fragment size is about 570bp, which is consistent with the target fragment size, and the yqaB gene fragment is recovered.

[0100] 2. Using the Escherichia coli K12 genome as a template, the primer pair P3 and P4 were used for PCR amplification, and the coding gene (rhaA) of L-rhamnose isomerase was obtained by PCR amplification. The PCR conditions were as follows: 98°C, 2min; 98°C, 20sec, 55°C, 20sec, 72°C, 45sec (30 cycles); 72°C, 5min. Detected by 1% agarose gel ele...

Embodiment 2

[0114] Embodiment 2, construct the recombinant plasmid that co-expresses methylglyoxal synthase and glycerol dehydrogenase

[0115] 1. Construction of recombinant plasmid pFU54

[0116] (1) Using the Escherichia coli K12 genome as a template, the primer pair P9 and P10 were used for PCR amplification, and the coding gene (gldA) of glycerol dehydrogenase was obtained by PCR amplification. The PCR conditions were as follows: 98°C, 2min; 98°C, 20sec, 55°C, 20sec, 72°C, 45sec (30 cycles); 72°C, 5min. Detected by 1% agarose gel electrophoresis, the fragment size was about 1100bp, which was consistent with the target fragment size, and the gldA gene fragment was recovered.

[0117] (2) After the pBAD vector was double-digested with XhoI and EcoRI, a large vector fragment with a size of about 4000 bp was recovered.

[0118] (4) The gldA gene fragment recovered in step (1) and the large vector fragment were used Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, ...

Embodiment 3

[0133] Embodiment 3, construct the host bacterium AODT that produces L-rhamnose

[0134] Chromosome editing of wild-type Escherichia coli K12 to knock out the aldehyde dehydrogenase A gene (aldA), L-1,2-propanediol oxidoreductase gene (fucO), and NADPH-dependent aldehyde reductase gene of wild-type Escherichia coli K12 (yqhD) and triose phosphate isomerase gene (tpiA), to obtain Escherichia coli mutant AODT. In this example, the P1 phage-mediated transfection method was used to construct the Escherichia coli mutant AODT, and the specific steps were as follows:

[0135] (1) Obtain the P1 of the donor bacteria: the donor bacteria BW25113△aldA::Kan (National Institute of Genetics (NIG, Japan), NIG number is JW1412), BW25113△fucO::Kan (National Institute of Genetics (NIG, Japan), NIG number JW2770), BW25113△yqhD::Kan (National Institute of Genetics (NIG, Japan), NIG number JW2978) and BW25113△tpiA::Kan (National Institute of Genetics (NIG , Japan), NIG No. JW3890) were inoculate...

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Abstract

The invention discloses an engineering bacterium producing L-rhamnose, a construction method and application thereof. The present invention enhances the synthesis pathway of L-rhamnose by overexpressing genes related to L-rhamnose synthesis in Escherichia coli, and at the same time increases the accumulation of precursor L-lactaldehyde by blocking the bypass pathway, A set of high-efficiency whole-cell catalytic synthesis of free L-rhamnose was developed, and the synthesis of L-rhamnose with glucose as a single substrate was realized for the first time in Escherichia coli. In this way, L-rhamnose can be catalyzed and efficiently synthesized by whole cells of Escherichia coli with cheap glucose as raw material. Not only is the synthesized rhamnose in a free state, which avoids complicated hydrolysis steps, but can be directly used for separation and purification, and can be obtained The product does not contain other monosaccharides or proteins, which reduces the burden of subsequent separation and purification.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a genetically engineered bacterium that uses glucose as a single substrate to produce L-rhamnose and its construction method and application. Background technique [0002] L-rhamnose is a methyl pentose, a derivative of L-mannose, also known as 6-deoxy-L-mannose. It widely exists in plant polysaccharides, glycosides, plant gums and bacterial polysaccharides in nature, and its sweetness is 33% of that of sucrose. Rhamnose is widely used in industrial production and scientific research. For example, it can be used to determine the permeability of the intestinal tract, as an intermediate in the synthesis of organic substances to synthesize spices, Furaneol, and synthetic cardiotonic drugs. It can also be directly used as a food additive to add to high-grade coffee, beverages, and meat products. With the deepening of research and development work, the application range of rhamno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P19/02C12N15/60C12N15/61C12N15/55C12N15/53
CPCC12N9/0006C12N9/16C12N9/88C12N9/90C12N15/70C12P19/02C12Y101/01006C12Y401/02019C12Y402/03003C12Y503/01014
Inventor 林白雪张莎莎史从容陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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