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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A direct liquid-culture (DLC) screening method has been developed which significantly reduces the time required for polyethylene glycol (PEG)-mediated protoplast transformation of the filamentous fungus, Aspergillus oryzae, when employed for evaluation of the secretory production of heterologous proteins. This method dramatically increases the throughput of the evaluation protocol.

Abstract

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

Introduction

Aspergillus oryzae is an important microorganism in the Japanese food industry that has been used for over 1,000 years in the production of fermented foods, such as sake (rice wine), shoyu (soy sauce), and miso (soybean paste)1,2. It has the ability to secrete a large amount of proteins, such as proteases and amylases3. Genome sequence information for A. oryzae is also available4. Moreover, powerful and practically useful genetic engineering techniques have been established for this fungus5,6,7,8,9,10. Favorable transformants have been used as hosts for secretory production of heterologous proteins11,12,13,14.

Electroporation, Agrobacterium-mediated transformation, and polyethylene glycol (PEG)-mediated protoplast transformation are the techniques used for introducing heterologous genes into A. oryzae15,16,17. The PEG-mediated protoplast transformation method has been widely used since it was first reported for Neurospora crassa in 197918. In this method, protoplasts are prepared and mixed with PEG and the heterologous gene that is to be introduced into the cells. The walls of protoplasts are enzymatically compromised, which makes the cells vulnerable to physical stress and changes in osmotic pressure19,20. The conventional screening method employed in the secretory production of heterologous proteins includes three steps: acquisition of positive transformants (on soft agar plate), selection of true and false transformants (on agar plate), and evaluation of the secretory production of heterologous proteins (in liquid culture); each of these steps takes seven days (Figure 1). Thus, the conventional method typically requires about three weeks.

The time required for screening of transformed A. oryzae cells is much longer than that required for other microorganisms commonly used in biotechnology research, making the process more cumbersome. For example, when using Escherichia coli as a host, it takes approximately two days from the introduction of DNA to confirmation of its effect21.

To circumvent the limitation associated with the use of A. oryzae as mentioned above, herein, a new direct liquid-culture (DLC) screening method is introduced, which enables a more rapid and simple screening for evaluation of the secretory production of heterologous proteins (Figure 1). In the DLC method, a uridine auxotrophic mutant and a nutrient-rich liquid medium are used. Using this method, the screening step can be completed within six days after the introduction of DNA into the protoplasts in a 200 mL flask format or within 10 days in a 24 well microplate format. Furthermore, the time-consuming and laborious preparation of agar plate media is not needed in this method. There is a huge advantage in using the newly described method, especially considering the fact that the conventional method requires two different media: the soft agar plate for acquisition of positive transformants, which requires careful handling and temperature control, and solid agar plate for selection of true transformants.

figure-introduction-3988
Figure 1: Schematic of polyethylene glycol (PEG)-mediated protoplast transformation of the filamentous fungus, Aspergillus oryzae.
(Top panel) Conventional screening method. (Bottom panel) Direct liquid-culture (DLC) screening method. Please click here to view a larger version of this figure.

Protocol

1. Preparation of fresh spore suspension

  1. Inoculate 20 μL of a stock spore suspension (1 x 107 spores/mL) in the center of a culture plate containing Czapek-Dox (CD) medium with 20 mM uridine (autoclaved at 121 °C for 20 min, Table 1).
  2. Incubate at 30 °C for 7 days to promote spore formation.
  3. Add 1.5 mL of 0.01 % Tween 20 solution (autoclaved at 121 °C for 20 min) to the culture plate containing spores and suspend the spores by scraping with a cell spreader.
  4. Collect the spore suspension in a sterile 1.5 mL microcentrifuge tube using a pipette.
  5. Store the suspension at 4 °C until use.
    NOTE: Use the spore suspension in the following procedure within 2 weeks. To make a stock for long-term storage, add glycerol to the spore suspension to a final concentration of 15% and store at -80 °C.

2. Preculture

  1. Add 100 mL of polypeptone-dextrin (PD) medium (Table 2) in a 500 mL Erlenmeyer flask, and autoclave it at 121 °C for 20 min.
  2. After confirming that the temperature of the medium is reduced, add the uridine solution, sterilized by passing through a filter with a pore size of 0.22 μm, to a final concentration of 20 mM.
  3. Add 200 μL of the spore suspension (1 x 107 spores/mL) prepared in step 1.4, and incubate at 30 °C with shaking at 120 rpm for 36 h.

3. Preparation of protoplasts

  1. Harvest the fungal biomass prepared in step 2.3 on a glass filter with pore size of 30 μm (autoclaved at 121 °C for 20 min).
  2. Pour 100 mL of distilled water (DW) (autoclaved at 121 °C for 20 min) on the glass filter and stir several times with a spatula (autoclaved at 121 °C for 20 min).
  3. After washing with DW, pour 100 mL of sodium chloride (NaCl) buffer (autoclaved at 121 °C for 20 min, Table 3) on the glass filter and stir several times with a spatula.
  4. Transfer about 1−2 mL (wet volume) of the cells to a 50 mL conical tube using a spatula (autoclaved at 121 °C for 20 min).
  5. After adding 15 mL of the enzyme solution (sterilized by passing through a 0.22 μm pore sized filter, Table 4) to a 50 mL conical tube, add NaCl buffer until the total volume reaches 30 mL.
  6. Close the lid and seal with paraffin film. Incubate at 30 °C with shaking at 60 rpm for 2 h.
  7. Flow the resulting solution through a 70 μm cell strainer attached to the 50 mL conical tube to remove the unreacted mycelia.
  8. Centrifuge the protoplast solution in a 50 mL conical tube at 2,150 x g and 4 °C for 20 min.
  9. Gently discard the supernatant to prevent pellet from dislodging. Add 1 mL of ice-cold sterilized solution B (autoclaved at 121 °C for 20 min, Table 5) and gently suspend the precipitated protoplasts by pipetting.
  10. Transfer the suspended protoplasts to a sterile 1.5 mL tube.
  11. Centrifuge at 2,220 x g and 4 °C for 5 min and remove the supernatant. Then, add 1 mL of ice-cold solution B and gently suspend the pellet by pipetting.
  12. Repeat step 3.9.
  13. Using a hemocytometer, measure the number of protoplasts under a microscope.
  14. Prepare a protoplast suspension (1−3 x 107 protoplasts/mL) in solution B and store it on ice until use.
    NOTE: Use the protoplast suspension immediately in the following procedure.

4. Introduction of DNA for secretory production of protein

  1. Using a plasmid containing a DNA cassette for secretory production of protein as a template, amplify the DNA fragment required for secretory production by PCR.
  2. Prepare the DNA sample to be used for transformation by processing the PCR product using a PCR purification kit.
  3. Add 1 mL of the protoplast solution, 200 μL of solution C (autoclaved at 121 °C for 20 min, Table 6), and 50 μL of the DNA sample (0.5−1 μg/μL) in a sterile, precooled 15 mL conical tube, and mix the solution gently by pipetting.
    NOTE: Solution C is highly viscous and can be difficult to handle; ensure thorough mixing by pipetting.
  4. Incubate the tube on ice for 30 min.
  5. Add 1.5 mL of solution C, mix gently by pipetting, and then, leave the solution at room temperature for 20−30 min.

5. Direct liquid culture screening

NOTE: Select the culture system using Erlenmeyer flasks (section 5.1) or microplates (section 5.2).

  1. Culture system using a 200 mL Erlenmeyer flask
    1. Add 50 mL of PD medium containing 0.8 M sorbitol to a 200 mL Erlenmeyer flask, and sterilize by autoclaving at 121 °C for 20 min.
    2. After confirming that the temperature of the medium is reduced, add the protoplast suspension prepared in step 4.5 to a final concentration of 1 x 105 protoplasts/mL.
    3. Incubate the culture for 6 days at 30 °C with shaking at 120 rpm.
    4. Collect 1 mL of the medium and store in a 1.5 mL tube at 4 °C. Store the culture sample at -20 ° C, if not analyzed immediately.
    5. Mix 20 μL of culture sample and 20 μL of sodium dodecyl sulfate (SDS) sample buffer (Table 7) in a 1.5 mL tube and boil at 95 ° C for 5 min.
    6. After cooling on ice, load 20 μL of the boiled sample and 5 μL of prestained protein standard on a precast gel and analyze the secretory production of the target heterologous protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)10.
  2. Culture system using a 24 well microplate
    1. Add the protoplast suspension prepared in step 4.5 to sterilized 0.8 M sorbitol-containing PD medium to a final concentration of 1 x 105 protoplasts/mL, and gently mix by pipetting.
    2. Add 1 mL of the solution prepared in step 5.2.1 to three wells in a 24 well microplate.
    3. Affix the lid and incubate at 30 °C with shaking at 175 rpm for 10 days.
    4. Collect 1 mL of medium and store in a 1.5 mL tube at 4 ° C. Store the culture sample at -20 ° C, if not analyzed immediately.
    5. Mix 20 μL of the culture sample and 20 μL of SDS sample buffer in a 1.5 mL tube and boil at 95 ° C for 5 min.
    6. After cooling on ice, load 20 μL of the boiled sample and 5 μL of prestained protein standard on a precast gel and analyze the secretory production of the target heterologous protein by SDS-PAGE10.

Results

The results for the introduction of the DNA expression cassette coding for Talaromyces cellulolyticus cellobiohydrolase (CBH: GenBank Accession Number E39854) into a uridine auxotroph of A. oryzae strain HO422 and screening for the secretory production of the heterologous protein are described below.

Preparation of fresh spore suspension

The final yield of spore suspension from one agar plate was 1 mL (1 x 1...

Discussion

We have developed a system that allows the screening of A. oryzae transformants more rapidly than the conventional method, by conducting liquid culture of the protoplasts. The most critical aspect of this method is the osmotic pressure of the liquid medium. The osmotic pressure suitable for liquid culture was optimized using sorbitol. The growth of A. oryzae strain HO4 was most active in the presence of 0.8 M sorbitol (Figure 3A). In the conventional method using soft agar ...

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors acknowledge Rinkei Okano for help with the experiments. The authors thank Professor Katsuya Gomi of Tohoku University and Professor Masayuki Machida of Kanazawa Institute of Technology for valuable discussions. This work was funded by Honda Research Institute Japan Co., Ltd.

Materials

NameCompanyCatalog NumberComments
1 M NaOHNACALAI TESQUE, INC.37421-05
1 M Tris-HCl (pH 7.5)FUJIFILM Wako Pure Chemical Corporation318-90225
1 M Tris-HCl (pH 6.8)FUJIFILM Wako Pure Chemical Corporation2106-100
1.5-mL Microcentrifuge tubeAS ONE Corporation1-1600-03
15-mL Conical centrifuge tubeBecton, Dickinson and Company352196
2-mercaptoethanolBio-Rad Laboratories, Inc.1610710
24-well MicroplateAGC TECHNO GLASS CO., LTD.3820-024
50-mL Conical centrifuge tubeBecton, Dickinson and Company352070
70-µm Cell strainerBecton, Dickinson and Company352350
AgarFUJIFILM Wako Pure Chemical Corporation010-15815
AutoclaveTOMY SEIKO CO.,LTD.LSX-700
Bromophenol blueFUJIFILM Wako Pure Chemical Corporation021-02911
CaCl2FUJIFILM Wako Pure Chemical Corporation038-24985
Casamino acidFUJIFILM Wako Pure Chemical Corporation393-02145
Cellulase R-10Cosmo Bio Co., Ltd.16419
Dextrin hydrateFUJIFILM Wako Pure Chemical Corporation044-00585
D-SorbitolFUJIFILM Wako Pure Chemical Corporation191-14735
e-PAGELATTO CORPORATIONE-T/R1020LUsed for precast gel in SDS-PAGE
Electrophoresis deviceATTO CORPORATIONWSE-1150Used for SDS-PAGE
FeSO4·7H2OFUJIFILM Wako Pure Chemical Corporation098-01085
Glass filter 17G3Tokyo Garasu Kikai Co., Ltd.0000094147
GlycerolFUJIFILM Wako Pure Chemical Corporation070-04941
HemocytometerFunakoshi Co., Ltd.521-10
High speed refrigerated centrifugeKUBOTA CORPORATION7780Used for centrifugation of samples in 50-mL conical centrifuge tubes
IncubatorTAITEC CORPORATION.G·BR-200Used for flask liquid culture and preparation of protoplasts
IncubatorTAITEC CORPORATION.BR-43FLUsed for microplate liquid culture and plate culture
KClFUJIFILM Wako Pure Chemical Corporation163-03545
KH2PO4NACALAI TESQUE, INC.28721-55
Lysing enzymeSigma-AldrichL1412-10G
MgSO4·7H2OFUJIFILM Wako Pure Chemical Corporation131-00405
Micro refrigerated centrifugeKUBOTA CORPORATION3740Used for centrifugation of samples in 1.5-mL microcentrifuge tubes
MicroscopeLeica MicrosystemsDMI6000 B
NaClFUJIFILM Wako Pure Chemical Corporation190-13921
NaH2PO4·2H2ONACALAI TESQUE, INC.31718-15
NaNO3FUJIFILM Wako Pure Chemical Corporation195-02545
Parafilm MBemis Company, IncPM-996
PCR Purification KitQIAGEN K.K28104
Petri dishSumitomo Bakelite Co., Ltd.MS-11900Used as culture plate
Polyethylene glycolSigma-AldrichP3640-500G
Polypeptone peptoneBecton, Dickinson and Company211910
Protein laddersBio-Rad Laboratories, Inc.161-0377Used as molecular weight marker in SDS-PAGE
Sodium dodecyl sulfateBio-Rad Laboratories, Inc.1610301
Sterile filterMerck KGaASLGP033RB
SucroseNACALAI TESQUE, INC.30404-45
Tween 20Tokyo Chemical Industry Co., Ltd.T0543
UridineSigma-AldrichU3750-25G
YatalaseTakara Bio Inc.T017

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Direct Liquid Culture DLC ScreeningAspergillus OryzaeHeterologous ProteinsProtoplast TransformationScreening Time ReductionPositive TransformantsSecretory ProductionNutrient rich MediumIndustrial ApplicationsProtein Production Protocol

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