Recombinant aspergillus terreus capable of high yielding itaconic acid, construction method for recombinant aspergillus terreus and application of recombinant aspergillus terreus

A construction method and technology of Aspergillus terreus, applied in the field of genetic engineering, can solve the problem that few people pay attention to the downstream pathway of itaconic acid, and achieve the effect of reducing metabolic burden, clear purpose, and realizing strengthening and accumulation.

Active Publication Date: 2020-12-04
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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  • Recombinant aspergillus terreus capable of high yielding itaconic acid, construction method for recombinant aspergillus terreus and application of recombinant aspergillus terreus
  • Recombinant aspergillus terreus capable of high yielding itaconic acid, construction method for recombinant aspergillus terreus and application of recombinant aspergillus terreus
  • Recombinant aspergillus terreus capable of high yielding itaconic acid, construction method for recombinant aspergillus terreus and application of recombinant aspergillus terreus

Examples

Experimental program
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Effect test

Embodiment 1

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

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

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

[0043] U-09973-F(5'-ccgacaacaagacctggatatac-3') and U-09973-R(hph2)(5'-ctttacgcttgcgatcccgaagaagactgctccttaatccacag-3') were used as primer pairs, and the genome of CICC40205 was used as a template for PCR amplification, 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 to 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 product was purif...

Embodiment 2

[0105] Example 2 Fermentation of recombinant Aspergillus terreus to produce itaconic acid.

[0106] 1. Shake flask screening of itaconic acid produced by recombinant Aspergillus terreus strains

[0107] Stable recombinant Aspergillus terreus 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 the mature spores into itaconic acid fermentation medium (55ml of itaconic acid fermentation medium in a 500ml Erlenmeyer flask), make three parallel bottles for each strain, and ferment for 72 hours at 37°C and 220rpm. The hyphae in the fermentation broth were removed by filtration, and the fermentation supernatant was appropriately diluted and analyzed by High Performance Liquid Chromatography (HPLC).

[0108] 2. Content and purity analysis of itaconic acid in fermentation broth

[0109] The fermentation supernatants of the recombinant strain and the original s...

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Abstract

The invention relates to recombinant aspergillus terreus capable of high yielding itaconic acid, a construction method for the recombinant aspergillus terreus and application of the recombinant aspergillus terreus and belongs to the technical field of genetic engineering. In order to increase the yield of synthesis of the itaconic acid, the invention provides the recombinant aspergillus terreus capable of high yielding the itaconic acid. The recombinant aspergillus terreus is obtained through mutating a monooxygenase gene of a cytochrome P450 of aspergillus terreus, wherein the monooxygenase gene of the cytochrome P450 is ATEG_09973 and has a nucleotide sequence shown in SEQ ID NO. 1. According to the recombinant strain provided by the invention, the itaconic acid fermentation yield is generally higher than that of an original strain, the itaconic acid fermentation yield of a strain with the highest yield in a shaking flask level is increased by about 17%-28% compared with that of theoriginal strain, and thus, the recombinant strain has a very high application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant Aspergillus terreus with high itaconic acid production and its construction method and application. Background technique [0002] Itaconic acid (itaconic acid) is considered to be one of the twelve bio-based chemicals with the most application prospects. It is mainly used as a monomer or comonomer in the chemical industry to synthesize acrylic fibers, resins, plastics, and rubbers. , coatings, surfactants and other polymer materials, and 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 market of itaconic acid huge, and it is increasing year by year. Industrially, itaconic acid is produced with good efficiency by the fermentation of Aspergillus terreus. However, compared with the similar Aspergillus niger fermented to produ...

Claims

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Application Information

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Patent Type & Authority Applications(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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