Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

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 fungus^5....

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 &#....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 M NaOH	NACALAI TESQUE, INC.	37421-05
1 M Tris-HCl (pH 7.5)	FUJIFILM Wako Pure Chemical Corporation	318-90225
1 M Tris-HCl (pH 6.8)	FUJIFILM Wako Pure Chemical Corporation	2106-100
1.5-mL Microcentrifuge tube	AS ONE Corporation	1-1600-03
15-mL Conical centrifuge tube	Becton, Dickinson and Company	352196
2-mercaptoethanol	Bio-Rad Laboratories, Inc.	1610710
24-well Microplate	AGC TECHNO GLASS CO., LTD.	3820-024
50-mL Conical centrifuge tube	Becton, Dickinson and Company	352070
70-µm Cell strainer	Becton, Dickinson and Company	352350
Agar	FUJIFILM Wako Pure Chemical Corporation	010-15815
Autoclave	TOMY SEIKO CO.,LTD.	LSX-700
Bromophenol blue	FUJIFILM Wako Pure Chemical Corporation	021-02911
CaCl2	FUJIFILM Wako Pure Chemical Corporation	038-24985
Casamino acid	FUJIFILM Wako Pure Chemical Corporation	393-02145
Cellulase R-10	Cosmo Bio Co., Ltd.	16419
Dextrin hydrate	FUJIFILM Wako Pure Chemical Corporation	044-00585
D-Sorbitol	FUJIFILM Wako Pure Chemical Corporation	191-14735
e-PAGEL	ATTO CORPORATION	E-T/R1020L	Used for precast gel in SDS-PAGE
Electrophoresis device	ATTO CORPORATION	WSE-1150	Used for SDS-PAGE
FeSO4·7H2O	FUJIFILM Wako Pure Chemical Corporation	098-01085
Glass filter 17G3	Tokyo Garasu Kikai Co., Ltd.	0000094147
Glycerol	FUJIFILM Wako Pure Chemical Corporation	070-04941
Hemocytometer	Funakoshi Co., Ltd.	521-10
High speed refrigerated centrifuge	KUBOTA CORPORATION	7780	Used for centrifugation of samples in 50-mL conical centrifuge tubes
Incubator	TAITEC CORPORATION.	G·BR-200	Used for flask liquid culture and preparation of protoplasts
Incubator	TAITEC CORPORATION.	BR-43FL	Used for microplate liquid culture and plate culture
KCl	FUJIFILM Wako Pure Chemical Corporation	163-03545
KH2PO4	NACALAI TESQUE, INC.	28721-55
Lysing enzyme	Sigma-Aldrich	L1412-10G
MgSO4·7H2O	FUJIFILM Wako Pure Chemical Corporation	131-00405
Micro refrigerated centrifuge	KUBOTA CORPORATION	3740	Used for centrifugation of samples in 1.5-mL microcentrifuge tubes
Microscope	Leica Microsystems	DMI6000 B
NaCl	FUJIFILM Wako Pure Chemical Corporation	190-13921
NaH2PO4·2H2O	NACALAI TESQUE, INC.	31718-15
NaNO3	FUJIFILM Wako Pure Chemical Corporation	195-02545
Parafilm M	Bemis Company, Inc	PM-996
PCR Purification Kit	QIAGEN K.K	28104
Petri dish	Sumitomo Bakelite Co., Ltd.	MS-11900	Used as culture plate
Polyethylene glycol	Sigma-Aldrich	P3640-500G
Polypeptone peptone	Becton, Dickinson and Company	211910
Protein ladders	Bio-Rad Laboratories, Inc.	161-0377	Used as molecular weight marker in SDS-PAGE
Sodium dodecyl sulfate	Bio-Rad Laboratories, Inc.	1610301
Sterile filter	Merck KGaA	SLGP033RB
Sucrose	NACALAI TESQUE, INC.	30404-45
Tween 20	Tokyo Chemical Industry Co., Ltd.	T0543
Uridine	Sigma-Aldrich	U3750-25G
Yatalase	Takara Bio Inc.	T017