Recombinant aspergillus terreus strain for producing itaconic acid as well as construction method and application of recombinant aspergillus terreus strain

A technology for producing itaconic acid and a construction method is applied in the field of genetic engineering to achieve the effects of strong application value, strong operability, and easy screening

Active Publication Date: 2020-11-17
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

[0004] In recent decades, attempts to increase the yield of itaconic acid have focused on traditional mutagenesis screening of high-yielding strains and optimization of fermentation processes, but none of them have resulted in a major breakthrough in the fermentation yield of itaconic acid.

Method used

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  • Recombinant aspergillus terreus strain for producing itaconic acid as well as construction method and application of recombinant aspergillus terreus strain
  • Recombinant aspergillus terreus strain for producing itaconic acid as well as construction method and application of recombinant aspergillus terreus strain
  • Recombinant aspergillus terreus strain for producing itaconic acid as well as construction method and application of recombinant aspergillus terreus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Construction of a recombinant Aspergillus terreus strain expressing trans-aconitic acid decarboxylase

[0050] 1. Construction of Aspergillus terreus trans-aconitic acid decarboxylase expression cassette: the trans-aconitic acid decarboxylase tad1 gene of the present invention is the trans-aconitic acid decarboxylase gene tad1 of smut zea zea, the trans-aconitic acid decarboxylase gene tad1 of coding The amino acid sequence of aconitic acid decarboxylase is shown in SEQ ID NO.1.

[0051] The trans-aconitic acid decarboxylase tad1 gene was artificially synthesized, and the codon was optimized for the expression of Aspergillus terreus. The optimized sequence is shown in SEQ ID No.2, and it was connected to the gene of Aspergillus nidulans glyceraldehyde triphosphate dehydrogenase to start PgpdAt and Aspergillus nidulans tryptophan synthase terminator TtrpC, cloned on the pUC57-Kan vector, constructed and obtained plasmid pmWM22( figure 2 ).

[0052] Using Pg...

Embodiment 2

[0066] Example 2. Using ptrA as a selection marker to construct a recombinant Aspergillus terreus strain expressing trans-aconitic acid decarboxylase

[0067] Repeat Example 1, the difference from Example 1 is that the selection marker gene used in the recombinant Aspergillus terreus strain described in this example is the pyrithione selection marker ptrA gene, and the specific method is described as follows:

[0068] 1. For the construction of the expression cassette of the trans-aconitic acid decarboxylase gene tad1 of Aspergillus terreus, refer to Example 1.

[0069] 2. Using the pyridinethiamine resistance gene ptrA as a selection marker to construct an Aspergillus terreus strain expressing tad1

[0070] 1) Trans-aconitic acid decarboxylase gene tad1 expression cassette and ptrA co-transformed Aspergillus terreus

[0071] Using ptrA-F (5'-gggcaattgattacgggatc-3') and ptrA-R (5'-atggggtgacgatgagccgc-3') as a primer pair, the pyrithione resistance gene was amplified by PCR ...

Embodiment 3

[0080] Example 3. Recombinant Aspergillus terreus fermented to produce itaconic acid

[0081] 1. Shake flask screening of itaconic acid produced by recombinant Aspergillus terreus strains

[0082] Stable recombinant Aspergillus terreus strains obtained in Examples 1 and 2 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).

[0083] 2. Content and purity analysis of itaconic acid in fermentation broth

[0084] The fermentation supernatants of the recombinant strain and the original ...

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Abstract

The invention discloses a recombinant aspergillus terreus strain for producing itaconic acid as well as a construction method and application of the recombinant aspergillus terreus strain, and belongsto the field of genetic engineering. In order to increase the yield of the itaconic acid, the invention provides the recombinant aspergillus terreus strain for producing the itaconic acid, and the recombinant aspergillus terreus strain is obtained by overexpression of trans-aconitate decarboxylase of ustilago maydis in an original strain. The original strain is aspergillus terreus. An amino acidsequence of the trans-aconitate decarboxylase is as shown in SEQ ID NO.1, and a nucleotide sequence of a gene for encoding the trans-aconitate decarboxylase is as shown in SEQ ID No.2. The itaconic acid fermentation yield of the recombinant strain provided by the invention is generally higher than that of the original strain, and the strain with the highest yield is improved by about 50% on the shake flask level compared with the original strain, so that the recombinant strain has very high application value.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant Aspergillus terreus strain producing itaconic acid, a construction method and application thereof. Background technique [0002] Itaconic acid is currently used as a monomer or comonomer in the polymer industry, mainly for the synthesis of acrylic fiber, resin, plastic, rubber, coating, surfactant, polymer chelating agent, papermaking, medicine , pesticides, light industry, food, silk and other fields, and have also been identified as metabolites with antibacterial activity produced in mammals. [0003] In Aspergillus terreus, the metabolic pathway of itaconic acid is shunted from the tricarboxylic acid cycle, and cis-aconitic acid in the tricarboxylic acid cycle is catalyzed by cis-aconitic acid decarboxylase (CAD) decarboxylation to form itaconic acid. Ustilago maydis is another microorganism that can naturally synthesize itaconic acid. In 2016, researchers fo...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12P7/44C12R1/66
CPCC12N9/88C12Y401/01C12N15/80C12P7/44C12N2800/22
Inventor 吕雪峰柴小高黄雪年周昌王敏刘璐谢义生周凯华
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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