Itaconic acid fermentation yield improvement bacterial strain, construction method thereof and itaconic acid production method using bacterial strain

A construction method, the technology of itaconic acid, applied in the field of genetic engineering, achieves the effect of strong application value, easy screening, and strong operability

Active Publication Date: 2014-06-04
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there has been no report on the improvement of itaconic acid fermentation yield by overexpressing aconitic acid decarboxylase in Aspergillus terreus

Method used

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  • Itaconic acid fermentation yield improvement bacterial strain, construction method thereof and itaconic acid production method using bacterial strain
  • Itaconic acid fermentation yield improvement bacterial strain, construction method thereof and itaconic acid production method using bacterial strain
  • Itaconic acid fermentation yield improvement bacterial strain, construction method thereof and itaconic acid production method using bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of Aspergillus terreus aconitic acid decarboxylase gene cad expression cassette

[0026] 1.1 Construction of expression vector pXH-1

[0027] With TtrpC-F (5'-cagacatgtagatctaagcttgcggccgcacttaacgttactgaaatc-3') and TtrpC-R (5'-gtcgtgccagtcgactctaga-3') as a primer pair, the plasmid pSGF957 (obtained by Seoul National university) was used to construct a plasmid map as follows figure 2 , Kim, J.G., Choi, Y.D., Chang, Y.J., Kim, S.U., Genetic transformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003, 25, 1509–1514) as a template for PCR amplification of the TtrpC fragment, and the product was subjected to Pci I (Fermentas, Catalog No.:#ER1871) and Xba I digestion and purification. pJJL-2 was obtained by cloning the PgpdAt1 promoter fragment into the pMD18-simple vector (Takara, Catalog No.: D103A), and then removing the Pci I restriction site on the pMD18-simple plasmid by site-directed mutagenesis (see image 3 ), its specific co...

Embodiment 2

[0055] Example 2 Preparation of recombinant Aspergillus terreus containing PgpdAt1-cad-TtrpC expression cassette

[0056] 2.1 Preparation of Aspergillus terreus CICC40205 protoplasts

[0057] 1) Inoculate the spore suspension of Aspergillus terreus CICC40205 into 50mL liquid medium ME (medium ME is 20g L per liter of water -1 Glucose, 2gL -1 NH 4 NO 3 , 20 mg L -1 (NH 4 ) 2 HPO 4 , 20 mg L -1 FeSO 4 , 0.4g L -1 MgSO 4 , 4.4 mg L -1 ZnSO 4 ), concentrated sulfuric acid to adjust the pH to 3.5, so that the spore concentration is about 10 7 cells / mL, cultured at 200rmp, 32°C for 12-18h.

[0058] 2) Use a sterile single layer of 200-mesh nylon cloth to collect the growing mycelium, and filter it with sterilized 0.6MMgSO 4 The solution was rinsed three times, pressed dry and placed in a sterile 50ml Erlenmeyer flask; weighed 1g of mycelium, added 10ml of enzymatic hydrolysis solution, and treated at 30°C, 60rpm for 1-3h. The composition of the enzymatic solution i...

Embodiment 3

[0064] Example 3 Fermentation of itaconic acid by recombinant Aspergillus terreus

[0065] 3.1 Shake flask screening of recombinant strains producing itaconic acid

[0066] 1) Preparation of itaconic acid fermentation medium IPM: 140g L -1 Glucose, 2gL -1 NH 4 NO 3 , 0.2gL -1 (NH 4 ) 2 HPO 4 , 20 mg L -1 FeSO 4 , 0.4g L -1 MgSO 4 , 40 mg L -1 ZnSO 4 , 40mgL -1 CuSO 4 , adjust the pH to 3.5 with sulfuric acid, and sterilize at 115°C for 10 minutes.

[0067] 2) Inoculate the recombinant strains obtained above and the stable recombinant Aspergillus terreus strains after passage of the recombinant strains into Aspergillus terreus sporulation slant medium (10g L -1 Glucose, 2gL -1 NaNO 3 , 0.2gL -1 ,KH 2 PO 4 , 20 mg L -1 FeSO 4 , 5g L -1 MgSO 4 , 0.5g L -1 NaCl, 40 mg L -1 ZnSO 4 , 40mgL -1 CuSO 4, 0.5% bran, 1.5% agarose, sterilized at 115°C for 15 minutes, then dispensed into test tubes, and sterilized at 115°C for 25 minutes to prepare s...

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Abstract

The invention relates to the field of gene engineering, and in particular relates to an itaconic acid fermentation yield improvement bacterial strain, a construction method thereof and an itaconic acid production method using the bacterial strain. The bacterial strain is recombinant aspergillus terreus obtained by inserting an aconitate decarboxylase gene in aspergillus terreus. A plasmid with a cis-aconitate decarboxylase gene expression box PgpdAt1-cad-TtrpC is constructed by gene engineering transformation, and then is inserted into the aspergillus terreus to catalyze cis-aconitate to obtain overexpression cis-aconitate decarboxylase to obtain the recombinant aspergillus terreus. The itaconic acid fermentation yield of the recombinant bacterial strain is generally higher than that of a starting bacterial strain.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a bacterial strain for improving the fermentation yield of itaconic acid and a method for constructing and utilizing the bacterial strain to produce itaconic acid. technical background [0002] Itaconic acid is an important chemical product, considered by the U.S. Department of Energy as one of twelve important high value-added chemicals, mainly used in acrylic fiber, resin, rubber, paint, paper, medicine, pesticide, light industry, food, silk and other fields. At present, all submerged fermentation factories producing itaconic acid at home and abroad use Aspergillus terreus. Such as figure 1 As shown, the metabolic pathway of itaconic acid in Aspergillus terreus is shunted from the tricarboxylic acid cycle, and cis-aconitic acid in the tricarboxylic acid cycle is in the process of cis-aconic acid decarboxylase (CAD) Catalytic decarboxylation to form itaconic acid (Kanamasa,...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/60C12N15/63C12P7/46C12R1/66
Inventor 李建军黄雪年吕雪峰李悦明张希铭李霞
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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