A kind of recombinant Aspergillus terreus producing itaconic acid and its construction method and application

A construction method and technology for producing itaconic acid, applied in the field of genetic engineering, can solve problems such as the downstream pathway of itaconic acid that few people pay attention to, and achieve the effect of reducing metabolic burden, strong operability, and clear purpose

Active Publication Date: 2022-07-05
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the vast majority of studies on the itaconic acid biosynthetic pathway of Aspergillus terreus are aimed at the itaconic acid biosynthetic pathway through genetic engineering transformation, such as figure 1 , while little attention has been paid to the downstream pathway of itaconic acid

Method used

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  • A kind of recombinant Aspergillus terreus producing itaconic acid and its construction method and application
  • A kind of recombinant Aspergillus terreus producing itaconic acid and its construction method and application
  • A kind of recombinant Aspergillus terreus producing itaconic acid and its construction method and application

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Embodiment 1

[0040] Example 1. Construction of knockout cytochrome P450 monooxygenase gene ATEG_09973 recombinant Aspergillus terreus

[0041] Attached below figure 2 The construction method of the recombinant Aspergillus terreus described in the present invention is described.

[0042] 1. Construction of the targeting element of the ATEG_09973 gene

[0043] Using U-09973-F(5'-ccgacaacaagacctggatatac-3') and U-09973-R(hph2)(5'-ctttacgcttgcgatcccgaagaagactgctccttaatccacag-3') as primer pair, PCR amplification was carried out with the genome of CICC40205 as template, and the product was purified After recovery, the upstream homology arm fragment of the ATEG_09973 gene was obtained; D-09973-F(hph2) (5'-ctgggttcgcaaagataattggtgctatcccgtcattcctagac-3') and D-09973-R (5'-cgagcactgaaggttaggtgcag-3') were used as primer pairs, and the The genome of CICC40205 was used as a template for PCR amplification, and the downstream homology arm fragment of the ATEG_09973 gene was obtained after the produ...

Embodiment 2

[0105] Example 2 Recombinant Aspergillus terreus fermented to produce itaconic acid.

[0106] 1. Shake flask screening for itaconic acid production by recombinant Aspergillus terreus strains

[0107] The recombinant strains of Aspergillus terreus, which were passaged and stable after the recombinant strains obtained in Example 1, were inoculated into Aspergillus terreus sporulation medium respectively, and cultured at 32° C. for 6 days to obtain mature spores. Then inoculate each spore cultured to maturity into itaconic acid fermentation medium (55ml of itaconic acid fermentation medium in a 500ml conical flask), and make three parallel flasks for each strain, ferment at 37°C and 220rpm for 72h. The mycelium in the fermentation broth was removed by filtration, and the fermentation supernatant was appropriately diluted and then analyzed by high performance liquid chromatography (HPLC).

[0108] 2. Analysis of the content and purity of itaconic acid in the fermentation broth

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Abstract

A recombinant Aspergillus terreus producing itaconic acid and a construction method and application thereof belong to the technical field of genetic engineering. In order to improve the synthetic yield of itaconic acid, the present invention provides a recombinant Aspergillus terreus that produces itaconic acid, which is obtained by mutating the cytochrome P450 monooxygenase gene in Aspergillus terreus. The oxygenase gene is ATEG_09973, and the nucleotide sequence is shown in SEQ ID NO.1. The itaconic acid fermentation yield of the recombinant strain provided by the invention is generally higher than that of the starting strain, and the strain with the highest yield is about 17%-28% higher than the starting strain at the shake flask level, and has strong application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant Aspergillus terreus producing itaconic acid and a construction method and application thereof. Background technique [0002] Itaconic acid is considered to be one of the twelve most promising bio-based chemicals. Currently, it is mainly used as a monomer or comonomer in the chemical industry to synthesize acrylic fiber, resin, plastic, and rubber. , coatings, surfactants and other polymer materials, at the same time, it is also widely used in papermaking, medicine, pesticides, light industry, food, construction and other fields. The good application prospect makes the demand for itaconic acid very large and increasing year by year. Industrially, itaconic acid is produced by fermentation of Aspergillus terreus with good efficiency. However, there is still a certain gap in the overall fermentation performance compared with the similar Aspergillus niger...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/90C12P7/44C12R1/66
CPCC12N9/0071C12N15/902C12P7/44
Inventor 吕雪峰柴小高王敏周昌黄雪年刘璐谢义生耿策周凯华
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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