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A kind of method for improving the yield of itaconic acid produced by fermentation of Aspergillus terreus

A technology of Aspergillus terreus and itaconic acid, which is applied in the field of bioengineering, can solve the problems of ATP excess and uncoupling, and achieve the effect of increasing production

Active Publication Date: 2020-02-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Aspergillus niger, due to the strengthening of the glycolytic pathway, excess NADH is synthesized, and if NADH is generated through the oxidative phosphorylation pathway to generate ATP to form NAD + , causing a surplus of ATP
Compared with the citric acid produced by Aspergillus niger, the pathway for the synthesis of itaconic acid by Aspergillus terreus only adds 2 steps, and both of them are reactions that do not require energy. Therefore, itaconic acid fermentation also faces NAD + Problems with pool replenishment and ATP formation uncoupling

Method used

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  • A kind of method for improving the yield of itaconic acid produced by fermentation of Aspergillus terreus
  • A kind of method for improving the yield of itaconic acid produced by fermentation of Aspergillus terreus
  • A kind of method for improving the yield of itaconic acid produced by fermentation of Aspergillus terreus

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: the extraction of Aspergillus niger RNA

[0033] Aspergillus niger spores were inoculated into citric acid fermentation medium, cultured at 35°C and 250 r / min for 48 hours, the balls were collected with mirocloth, washed 3 times with sterile ultrapure water, filtered to dry, and quickly frozen in liquid nitrogen. The tissue was thoroughly ground by grinding with liquid nitrogen, and the total RNA of Aspergillus niger was extracted by QIAGEN RNeasy Plant Mini Kit. The RNA was reverse-transcribed into cDNA with TAKARA PrimeScript RT reagent Kit with gDNA Eraser.

Embodiment 2

[0034] Embodiment 2: the extraction of Aspergillus niger genomic DNA

[0035] Inoculate Aspergillus niger spores into ME liquid medium (3% malt extract, 0.5% tryptone), culture at 250 r / min at 35°C for 48 hours, collect the balls with mirocloth, wash 3 times with sterile ultrapure water, and filter dry Moisture, quickly frozen in liquid nitrogen. The tissue was thoroughly ground by grinding with liquid nitrogen, and the filamentous fungal genome was extracted using the DNeasy Plant Mini Kit from QIAGEN.

Embodiment 3

[0036] Embodiment 3: Construction of AOX protein expression cassette

[0037]Use primers trp-F (sequence shown in SEQ ID NO.7) and trp-R (sequence shown in SEQ ID NO.8) to amplify the trp terminator with pAN7-1 as a template, and the upstream and downstream sequences contain Pst I and The Hin dIII site was ligated to pMD19 for sequencing, digested with these two restriction endonucleases, and ligated the sequence to pUC19 digested with the same restriction enzymes to obtain pUC19-trp. Use primers Pgas-F (sequence shown in SEQ ID NO.9) and Pgas-R (sequence shown in SEQ ID NO.10) to amplify the Pgas promoter from Aspergillus niger genomic DNA, and the sequence contains Eco RI and Kpn at both ends I enzyme cutting site, enzyme cutting, the sequence is connected to pUC19-trp which is also digested to obtain pUC-Pgas-trp. Use primers AOX-F (sequence shown in SEQ ID NO.11) and AOX-R (sequence shown in SEQ ID NO.12) to amplify the AOX gene from Aspergillus niger cDNA, and the two en...

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Abstract

The invention belongs to the technical field of bioengineering and discloses a method for increasing yield of itaconic acid produced by fermentation of aspergillus terreus. By adoption of acid-induced promoters derived from aspergillus niger, lateral respiratory chain protein AOX of the aspergillus niger is introduced into the aspergillus terreus to improve supply of an intracellular NAD+ bank, and accordingly glycolytic pathway can be enhanced to realize increase of itaconic acid yield. The itaconic acid yield of recombinant aspergillus terreus strains obtained by the method is increased by about 20% to reach 60g / L. The method provides a fresh idea for itaconic acid production.

Description

technical field [0001] The invention relates to a method for increasing the yield of itaconic acid produced by fermentation of Aspergillus terreus, belonging to the technical field of bioengineering. Background technique [0002] Itaconic acid is a five-carbon dicarboxylic acid containing a methylene group. This unsaturated bond endows itaconic acid with the function of polymerization reaction, so itaconic acid is widely used in the production of resins and synthetic fibers. In addition, Itaconic acid as a bioactive component can be used in agriculture and pharmaceutical industries, and it can be used as an initial compound for enzymatic conversion to synthesize a multifunctional platform compound. [0003] At present, the production of itaconic acid mainly relies on submerged aerobic fermentation using Aspergillus terreus. The metabolic pathway of itaconic acid produced by Aspergillus terreus is that glucose synthesizes citric acid through glycolysis pathway and TCA cycle,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/44C12N1/15C12N15/80C12R1/66
Inventor 刘龙殷娴陈坚堵国成李江华
Owner JIANGNAN UNIV