Method to produce protein in Aspergillus niger's resting spores using SSRNA

Patent metadata

Field	Value
Publication number	US-11286505-B2
Application number	US-201916296267-A
Country	US
Kind code	B2
Filing date	Mar 8, 2019
Priority date	Sep 9, 2016
Publication date	Mar 29, 2022
Grant date	Mar 29, 2022

Abstract

A method to produce protein in Aspergillus niger's sleeping spores using single-stranded RNA is provided. The method includes three steps: culture of Aspergillus niger and collection of spores, pretreatment of Aspergillus niger spores, and electroporation of Aspergillus niger spores using HDEN method. Non-germinated spores are used as a starting material for introduction of exogenous molecules. The exogenous protein coding single-stranded RNA is introduced into the resting spores of Aspergillus niger by employing the HDEN electrotransformation technique to express protein. This method is simple and fast, the effect is excellent, and the transformation rate reaches more than 90%.

First claim

I claim:

1. A method for direct transformation of exogenous single-stranded RNA into resting spores of Aspergillus niger to express protein, comprising the steps of:
A) culturing Aspergillus niger and collecting spores, as follows:
inoculating Aspergillus niger onto a surface of a solid agar medium, and culturing until Aspergillus niger spores are overgrown on the surface of the medium, washing the Aspergillus niger spores from the surface of the medium to create a suspension of spores, aspirating off and filtering the suspension of the spores to remove mycelia, collecting the filtrate containing the spores, and centrifuging the filtrate to collect pelleted resting spores;

B) pretreating the Aspergillus niger spores, as follows:
re-suspending the spores in an electroporation buffer, and centrifuging to collect spore pellets, repeating the re-suspension and centrifugation steps 3-4 times, and re-suspending the last collected spore pellets in the electroporation buffer, to obtain an Aspergillus niger spore suspension with a spore concentration of 10 4 -10 11 spores/ml,
wherein the electroporation buffer consists of 4-hydroxyethyl piperazineethanesulfonic acid having a final concentration of 0.01-100 mmol/L and mannitol having a final concentration of 0.5-5000 mmol/L, and the pH of the electroporation buffer is 3.0-9.5; and

C) electroporating the Aspergillus niger spores by using high-density distributed electrode network (HDEN) method, as follows:
adding the Aspergillus niger spore suspension prepared in step B) and a RNA to be transformed to wells of a cell culture plate and mixing uniformly, to obtain a mixture of the spores and the RNA, placing the cell culture plate on an ice bath for 10-15 min, subsequently electroporating using the HDEN method, by inserting an electroporator head fitted with a matrix electrode into the mixture of the spores and the RNA, and energizing, to generate an electric field inside the mixture of the spores and the RNA, placing the cell culture plate on the ice bath again for 10-15 min after electroporation, and subsequently aspirating off the mixture of the spores and the RNA, to obtain resting spores of Aspergillus niger with introduction of exogenous RNA,
wherein a ratio of the Aspergillus niger spore suspension to the RNA to be transformed is 0.6-60000 μl of Aspergillus niger spore suspension to 0.1-10000 μg of RNA;
wherein parameters for the electroporation comprise: a voltage of 1-6000 V, pulse duration of 2-2000000 ms, and repeat for 1-100 times at an interval of 5-50000 ms;
the RNA to be transformed is a single-stranded RNA encoding an exogenous protein; and

the RNA encodes green fluorescent protein, red fluorescent protein, or yellow fluorescent protein.

2. The method for direct transformation of exogenous single-stranded RNA into resting spores of Aspergillus niger to express protein according to claim 1 , wherein, the medium in the step A) is potato dextrose agar (PDA) medium, yeast extract-peptone-dextrose (YPD) medium, or Czapek-Dox medium.

3. The method for direct transformation of exogenous single-stranded RNA into resting spores of Aspergillus niger to express protein according to claim 1 , wherein, in the step A), Aspergillus niger is cultured at a temperature of 16-40° C. with 15-85% humidity for 3-15 days.

4. The method for direct transformation of exogenous single-stranded RNA into resting spores of Aspergillus niger to express protein according to claim 1 , wherein, in the step B), the Aspergillus niger spore suspension is observed under a microscope before electroporation, to confirm that the spore suspension is free of contamination with mycelia and the resting spores are non-germinated, and subsequently carrying out the electroporation.

5. The method for direct transformation of exogenous single-stranded RNA into resting spores of Aspergillus niger to express protein according to claim 1 , wherein, in the step C), in which the ratio of the Aspergillus niger spore suspension to the RNA to be transformed is 60 μl of Aspergillus niger spore suspension to 10 μg of RNA to be transformed.

6. The method for direct transformation of exogenous single-stranded RNA into resting spores of Aspergillus niger to express protein according to claim 1 , wherein, in the step C), the parameters for the electroporation comprise: a voltage of 400 V, pulse duration of 2000 ms, and repeat for 3 times at an interval of 500 ms.

Assignees

• Univ Fuzhou

Inventors

• Lin Jun

Classifications

• C12R2001/685

  Aspergillus niger · CPC title

• C12N15/87 Primary

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12N15/90 Primary

  Stable introduction of foreign DNA into chromosome · CPC title