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Method for improving production capacity of Aspergillus niger saccharifying enzyme and recombinant Aspergillus niger strain

A technology of Aspergillus niger strain and production capacity, applied in the field of bioengineering, can solve problems such as optimization of Aspergillus niger cell factory

Active Publication Date: 2021-07-02
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The research on Aspergillus niger is mainly carried out from the aspects of protein secretion pathway transformation, hyphal morphology, enhancement of acid production capacity, metabolic regulation, etc., but very few studies focus on fatty acid metabolism Optimizing the Aspergillus niger cell factory from angles

Method used

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  • Method for improving production capacity of Aspergillus niger saccharifying enzyme and recombinant Aspergillus niger strain
  • Method for improving production capacity of Aspergillus niger saccharifying enzyme and recombinant Aspergillus niger strain
  • Method for improving production capacity of Aspergillus niger saccharifying enzyme and recombinant Aspergillus niger strain

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Experimental program
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Effect test

Embodiment 1

[0063] The construction of embodiment 1 recombinant Aspergillus niger strain

[0064]1) Through basic molecular cloning steps such as PCR, one-step cloning, gel recovery, and plasmid purification, a plasmid is constructed exogenously to integrate the target gene and the plasmid vector. In this example, the Phanta high-fidelity enzyme PCR system is used to amplify the target gene to integrate the target gene and the plasmid The vector, PCR system and reaction conditions are shown in Table 3 and Table 4; the plasmid used is the expression plasmid initiated by the Tet-on system, which is inserted into the PmeI restriction site on the plasmid pFW22.1 containing the inducible expression of the Tet-on system For the coding region of the above-mentioned genes that need to be overexpressed, the plasmid construction strategy is as follows: figure 1 .

[0065] 2) Transform the recombinant plasmid obtained in step 1) into Escherichia coli DH5α competent, use Tag Mix enzyme to colony PCR...

Embodiment 2

[0084] Embodiment 2 Recombinant Aspergillus niger strain shaking flask fermentation experiment

[0085] The shake flask experiment was carried out using the overexpression strain constructed in Example 1, and the culture medium was a shake flask culture medium. The inoculum size is 10 6 spores / ml, the culture condition is 30°C, 250rpm culture for 72h, add 5 glass beads to prevent balling, and shake flask fermentation, add 10μg / ml inducer Doxycycline (DOX ), since DOX needs to be kept away from light, it may be inactivated if exposed to light for a long time, so add DOX every 12 hours. Samples were taken every 24 hours after inoculation, and the dry weight of the bacteria, total secreted protein and glucoamylase activity were measured. And about 26 hours in the mid-logarithmic growth phase, that is, 8 hours after induction, the mycelia were preserved for mRNA extraction. In another parallel experiment group, DOX was added from 42h and added every 12h.

[0086] The dry weigh...

Embodiment 3

[0091] Embodiment 3 Recombinant Aspergillus niger strain reactor culture experiment

[0092] Batch culture fermentation: The culture was carried out on 5L tanks with weighing. The liquid volume of the 5L tank is 3L, and the inoculation volume is 10 6 spores / ml, and the culture medium is the initial fermentation medium of the reactor. The stirring speed and ventilation speed of the 5L tank were 750rpm and 2vvm respectively, the tank pressure was 0.05MPa, and the temperature was 30°C. During the batch culture, samples were taken every two hours after addition of the inducer DOX for monitoring the growth curve. When the online monitoring curve CER, OUR began to decrease, dissolved oxygen increased and then transferred to chemostat culture. When CER and OUR begin to decrease, it proves that the fermentation is over. At this time, feed feeding is started and the chemical constant fermentation stage is entered. The tail carbon and tail oxygen concentrations in the fermentor were...

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Abstract

The invention discloses a method for improving production capacity of Aspergillus niger saccharifying enzyme and a recombinant Aspergillus niger strain. The method comprises the following steps: integrating uracil defective (pyrG-) strains with high yield of aspergillus niger saccharifying enzyme by using a Tet-on system through a promoter of an exogenous plasmid and utilizing homologous recombination, so that at least one of acyl-coenzyme A dehydrogenase coding genes, acetyl-coenzyme A acyltransferase coding genes or cytochrome P450 monooxygenase coding genes is over-expressed; and the saccharifying enzyme production capacity of the aspergillus niger is greatly improved by regulating and controlling a fatty acid metabolic pathway.

Description

technical field [0001] The application relates to the technical field of bioengineering, in particular to a method for improving the production capacity of Aspergillus niger glucoamylase and recombinant Aspergillus niger strains. Background technique [0002] Glucoamylase (Glucoamylase, E.C.3.2.1.3.), also known as glucoamylase (amyloglucosidase), is an acidic glycoproteinase with one peptide chain, which has exolytic activity and can hydrolyze starch, dextrin, glycogen and other carbohydrates The non-reducing end connected by 1,4 glycosidic bonds on the carbon chain, the final product is β-D-glucose, which can also hydrolyze α-1,6 and α-1,3 glycosidic bonds, but the hydrolysis speed is relatively slow. Glucoamylase is the main enzyme for hydrolyzing starch to produce glucose. Since the glucoamylase derived from Aspergillus niger was purified for the first time in the 1970s, it has been widely used in the industrial production of starch sugar, food, medicine, brewing, etc. ...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N9/34C12R1/685
CPCC07K14/38C12N9/2428
Inventor 欧阳立明祁洁隋雨菲肖贤尊庄英萍
Owner EAST CHINA UNIV OF SCI & TECH
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