Mutant blocked in glycerol oxidaion pathway for producing 1,3-propanediol

A technology of propylene glycol and glycerol, applied in biochemical equipment and methods, transferase, enzyme and other directions, can solve problems such as high manufacturing cost and waste oil generation

Active Publication Date: 2014-06-25
ACTIVON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, problems with such chemical methods are that in the production of 1,3-propanediol, they require high temperature or high pressure processes, thus resulting in high manufacturing costs and waste oils containing environmental pollutants
However, there is currently no prior art dealing with methods for reducing by-products that accumulate in large quantities when genetically engineered variants are used to manufacture 1,3 propanediol

Method used

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  • Mutant blocked in glycerol oxidaion pathway for producing 1,3-propanediol
  • Mutant blocked in glycerol oxidaion pathway for producing 1,3-propanediol
  • Mutant blocked in glycerol oxidaion pathway for producing 1,3-propanediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of recombinant bacterial strains blocked by the oxidation-reduction metabolic pathway of glycerol

[0055]For the redesign of the glycerol metabolic pathway, the recombinant bacterial strain in which the glycerol metabolic pathway in Klebsiella MGH78578 (ATCC 700721) was completely blocked was prepared as the basic strain. The antibiotic resistance profile of the Klebsiella MGH 78578 strain was analyzed in order to fix a selection marker that could be used in the genetic recombination process. Therefore, it was found that apramycin (50 / ml) can be used as an antibiotic marker for the screening of recombinant strains. In addition, the plasmid initially present in the strain was removed by processing and tetracycline was added as an available selectable marker. Using plasmid DNA-treated Klebsiella MGH 78578 strain (named "Cu") as a parent strain, the DhaD gene of the DhaB enzyme reactivator, DhaT gene, DhaR regulator, and dha regulator ( figure 2 ...

Embodiment 2

[0077] Example 2: Analysis of the properties of Klebsiella strains AK and AR

[0078] The proliferation and glycerol metabolism properties of recombinant Klebsiella strains AK and AR, in which the anaerobic metabolic pathway for glycerol was removed, were analyzed. Each strain of AK and AR was cultured into a medium supplemented with glycerol and incubated at 37° C. at 180 rpm for 12 hours. Then, the degree of proliferation of the cells was studied, and the content of glycerol present in the culture supernatant and the metabolites produced including 1,3-propanediol were analyzed by chromatography (device used: Agilent 1200 (refractive index detector , RID); used column: Aminex HPX-87H (Bio-Rad) 300mm×78mm; used solvent: 65:35 deionized water-acetonitrile (0.005M H 2 SO 4 ); and flow rate: 0.5ml / min. The AK and AR strains showed cell proliferation rates that were about two times slower than the parent strain Cu used as a control, and their glycerol consumption rates and 1,3-...

Embodiment 3

[0079] Embodiment 3: the bacterial strain that the reductive pathway of preparation glycerol is repaired

[0080] (1) Preparation of plasmid DNA for repairing the reduction pathway of glycerol

[0081] DhaB reactivase gene (orfW)-orfX DNA fragments and dhaT, yqhD (from E. coli) or yqhD homologous genes (from Klebsiella) with 1,3-propanediol oxidoreductase activity were amplified using the following primer sequences. The amplified gene was cloned into pGEM TEasy vector, and its basic sequence was analyzed. Then, if Figure 5 Plasmid DNAs were prepared as indicated in .

[0082] SEQ ID NO: 9: 5'-AGATCTATGAGCTATCGTATGTTTGA-3'(dhaT-BglII F)

[0083] Sequence number 10: 5'-CTCGAGAAGCTTCAGAATGCCTGGCGGAAAAT-3'

[0084] (dhaT-HindIII / XhoI R)

[0085] SEQ ID NO: 11: 5'-AGATCTATGAACAACTTTAATCTGCAC-3'(yqhD-BglII F)

[0086] SEQ ID NO: 12: 5'-AGATCTATGAATAATTTCGACCTGCA-3'(yqhD-HindIII /

[0087] XhoI R)

[0088] SEQ ID NO: 13: 5'-AGATCTATGAATAATTTCGACCTGCA-3'(yqhD Kle BglII F)

[...

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Abstract

The present invention relates to a microbial mutant in which the gene encoding transcription activator or the gene encoding dihydroxyacetone kinase in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source is deleted or inactivated and to a method for producing 1,3- propanediol using the mutant. When the mutant according to the present invention is used, the production of byproducts which are inevitably obtained when producing 1,3 -propanediol from glycerol using prior strains can be minimized, and thus the cost of purification of 1,3-propandiol can be reduced.

Description

technical field [0001] The present invention relates to a microbial mutant in which a genetically encoded transcriptional activator or a genetically encoded dihydroxyacetone kinase is removed or inactivated in a microorganism capable of using glycerol as a carbon source to produce 1,3-propanediol, It also relates to a method for producing 1,3-propanediol using the mutant. Background technique [0002] 1,3-Propanediol can be used as a raw material for the synthesis of polyesters, polyethers, polyurethanes, etc., and is used in a variety of applications, including fibers such as high-performance clothing, blankets or automotive fabrics and plastic films. In particular, polytrimethylene terephthalate (PTT), produced by the polymerization of 1,3-propanediol and terephthalic acid, has excellent physical properties and a melting point of 228 lower than that of polyethylene terephthalate (PET). °C melting point. Therefore, polytrimethylene terephthalate (PTT) has high practicabil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21
CPCC12Y207/01029C12N9/1205C12P7/18
Inventor 金哲镐徐正又许仙宴徐美渶吴白禄白珍晤徐必守崔珉镐
Owner ACTIVON
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