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Engineering bacteria for producing L-rhamnose, and construction method and application of engineering bacteria

A technology of rhamnose isomerase and genetically engineered bacteria, applied in the field of genetic engineering, can solve problems such as low yield, affecting rhamnose yield and yield, and many by-products

Active Publication Date: 2019-12-03
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 for producing L-rhamnose, and construction method and application of engineering bacteria
  • Engineering bacteria for producing L-rhamnose, and construction method and application of engineering bacteria
  • Engineering bacteria for producing L-rhamnose, and construction method and application of engineering bacteria

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 engineering bacteria for producing L-rhamnose, and a construction method and application of the engineering bacteria. Genes associated with synthesis of the L-rhamnose are over-expressed in escherichia coli, a synthesis pathway of the L-rhamnose is enhanced, meanwhile, through blocking of a bypass path, the accumulation amount of a precursor L-lactic aldehyde is increased,a set of efficient whole-cell catalytic synthesis method of free L-rhamnose is developed, and the L-rhamnose is synthesized firstly in the escherichia coli by taking glucose as a single substrate. Inthis way, the cheap glucose can serve as raw materials, the L-rhamnose is efficiently synthesized through whole-cell catalysis of the escherichia coli, the synthesized rhamnose is in a free state, thecomplex hydrolysis step is avoided, the synthesized rhamnose can be directly used for separation purification, an obtained product does not contain other monosaccharides or proteins either, and thusthe burden of subsequent separation purification is reduced.

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 and added to high-grade coffee, beverages, and meat products. With the deepening of research and development work, the application range of rha...

Claims

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Application Information

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Patent Type & Authority Applications(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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