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Construction method and use of fumaric acid producing candida glabrata engineering strain

A technology of Torulopsis glabrata and fumaric acid, which is applied in the field of fermentation engineering and can solve problems such as difficulty in dissolving oxygen and increasing the viscosity of fermentation broth

Inactive Publication Date: 2011-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there are many advantages in the production of fumaric acid by fermentation, the fermentation method of Rhizopus oryzae is mostly used in the production of fumaric acid at present. , causing difficulty in dissolving oxygen, etc.

Method used

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  • Construction method and use of fumaric acid producing candida glabrata engineering strain

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Embodiment 1

[0024] Example 1 Construction of Toruula glabrata engineering bacteria producing fumaric acid

[0025] 1) Construct the free expression vector pY26 TEF-RoFUM1-GPD-RoMDH

[0026] The RoMDH gene that has been connected into the pMD18 T-vector was double-digested, recovered and purified by restriction endonucleases BamHI and HindIII, and connected to the constructed free expression vector pY26 TEF-RoFUM1 (overnight at 16°C) for further construction Episomal expression vector pY26 TEF-RoFUM1-GPD-RoMDH.

[0027] 2) Convert step 1) to connect the product

[0028] After the ligation product was transformed into JM109 competent cells, the LB plate with ampicillin resistance was spread and cultured overnight at 37°C.

[0029] 3) Extraction plasmid digestion verification

[0030] The transformants were picked to extract plasmids for enzyme digestion verification, and all positive clones were found, indicating that the free expression vector pY26 TEF-RoFUM1-GPD-RoMDH had been successf...

Embodiment 2

[0034] Embodiment 2 utilizes Torulopsis glabrata engineered bacterium to produce fumaric acid

[0035] Pipette 200 μL of Torulopsis glabrata engineering bacteria into the seed medium (20mL / 200mL Erlenmeyer flask) from the glycerol tube, shake the flask at 30°C and 200r / min for 24h, and then inoculum with 10% inoculum (v / v) Access to the fermentation medium. Shake flask fermentation: 50mL of fermentation medium in a 500mL Erlenmeyer flask, the temperature is 30°C, the rotation speed is 200r / min, and the fermentation time is 60h. Adopt high-performance liquid chromatography (HPLC) to measure: the output of fumaric acid is 35mg / L, is 6 times of starting bacterial strain.

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Abstract

The invention discloses a construction method and use of a fumaric acid producing T.glabrata candida glabrata engineering strain and belongs to the field of fermentation engineering. In the invention, a recombinant T.glabrata FMME045 strain is obtained by overexpression of a malic dehydrogenase (RoMDH) gene and a fumarase (RoFUM1) gene from rhizopus oryzae in a uracil deficient (Ura) T.glabratadeltaura3 strain of a pyruvic acid producing strain T.glabrata in a free expression mode. When the strain is used for fermentation production of fumaric acid, the fumaric acid yield after the fermentation is performed for 60 hours is increased by 6 times to 35mg / L compared with that of a starting strain. Thus, a new approach is provided for producing fumaric acid by a microbial fermentation process. The method has a bright application prospect.

Description

technical field [0001] The invention relates to a construction method and application of a strain of Toruula glabrata engineering bacteria producing fumaric acid. By constructing a heterologous fumaric acid production pathway in the cytoplasm of the uracil (Ura)-deficient T. glabrataΔura3 of the pyruvate-producing strain T. glabrata, the carbon metabolic flux was redistributed and the accumulation of fumaric acid was promoted. , belonging to the field of fermentation engineering. Background technique [0002] Fumaric acid, also known as Fumarate or Trans-Butenedioic Acid, is considered to be one of the top ten framework substances obtained from glucose as a raw material through biochemical transformation. It is an important intermediate metabolite in the TCA cycle of organisms, and it is also an important organic chemical raw material and fine chemical product. Due to its non-toxic and low corrosion characteristics, it is widely used in food, medicine, chemical industry, p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/53C12N15/63C12P7/46C12R1/645
Inventor 刘立明陈修来杨松鑫陈坚
Owner JIANGNAN UNIV
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