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Method for increasing yield of itaconic acid produced by fermentation of aspergillus terreus

A technology of Aspergillus terreus and itaconic acid, applied in the field of bioengineering, can solve problems such as excess ATP and uncoupling, and achieve the effect of increasing yield

Active Publication Date: 2016-01-27
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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  • Method for increasing yield of itaconic acid produced by fermentation of aspergillus terreus
  • Method for increasing yield of itaconic acid produced by fermentation of aspergillus terreus
  • Method for increasing yield of itaconic acid produced by fermentation of aspergillus terreus

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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 RNeasyPlantMiniKit. The RNA was reverse-transcribed into cDNA with PrimeScriptRTreagentKitwithgDNAEraser from TAKARA Company.

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 by QIAGEN DNeasyPlantMiniKit.

Embodiment 3

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

[0037]Use primers trp-F (sequence shown in SEQIDNO.7) and trp-R (sequence shown in SEQIDNO.8) to amplify the trp terminator with pAN7-1 as a template, the sequence contains PstI and HindIII sites upstream and downstream, ligated Sequencing was performed on pMD19, digested with these two restriction endonucleases, and the sequence was ligated to pUC19 digested with the same restriction enzymes to obtain pUC19-trp. Utilize primers Pgas-F (sequence shown in SEQIDNO.9) and Pgas-R (sequence as shown in SEQIDNO.10) to amplify the Pgas promoter from Aspergillus niger genomic DNA, the two ends of the sequence contain EcoRI and KpnI restriction sites, Digested with restriction enzymes, and ligated the sequence to pUC19-trp which was also digested with restriction enzymes to obtain pUC-Pgas-trp. Utilize primer AOX-F (sequence as shown in SEQIDNO.11) and AOX-R (sequence as shown in SEQIDNO.12) to amplify AOX gene fro...

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