Financial support: Thai Research Fund, Graduate School of Kasetsart University.

Keywords: biolistic, cosmid, hygromycin, Monascus purpureus, transformation.

Present address: #Mission College, Muak Lek Campus, 195 Moo 3, Tambon Muak Lek, Muak Lek District, Saraburi Province 18180, Thailand. Tel: 66 36 344777, Fax: 66 36 341629.

Albino strain KB20M1 of Monascus purpureus was genetically transformed to hygromycin B resistance with cosmid pMOcosX, using biolistic bombardment. Conidia and mycelial fragments were used as the recipient material and rupture disk strengths of 6.2 and 9.3 MPa each yielded a transformed isolate from five bombardments. Southern analysis suggested a single copy of the cosmid had integrated into each of the transformants. Both of the independent transformants formed cleistothecia but ascospore formation was greatly reduced or absent, suggesting the integration and expression of the genes carried on pMOcosX interfered with fertilization and/or ascospore formation in this homothallic fungus.

Monascus purpureus is a homothallic fungus found on red rice (Anka or Ang-kak). It has been used as a natural food colorant and traditional medicine in Asia for centuries (Chu and Poon, 1993) and has been used as a substitute for nitrite in meat preservation (Fink-Gremmels et al. 1991). Several studies have focused on optimizing pigment production for use in foods (Chu and Poon, 1993; Berset et al.1995; Teng and Feldheim, 2000; Teng and Feldheim, 2001). The fungus also is of interest from a medical standpoint because it produces monacolin K, an inhibitor of cholesterol biosynthesis (Ma et al.2000). Hence, numerous genes in this fungus are of potential interest from a food and medicine point of view. Studies of genes are greatly facilitated by the availability of a genetic transformation system, but genetic transformation has not been reported in M. purpureus. This study was undertaken to develop a simple and robust genetic transformation system applicable to genetic study of M. purpureus.

Microorganism and cultivation

The microorganism used was Monascus purpureus. UV-induced albino mutant KB20M1 (Yongsmith et al. 2000) was used as the recipient in fungal transformations. Cultures were maintained on modified complete medium (CM) containing 0.5% sucrose, 0.6% yeast extract, and 0.6% casein hydrolysate at 28ºC in the dark.

Growth inhibition of KB20M1 by hygromycin B was determined prior to transformation experiments by inoculation of the fungus to CM containing 6.25, 12.5, 25, 50, 100 or 200 µg/ml hygromycin B. Ten replications were incubated at 28ºC in the dark and growth rates recorded after 14 d.

Biolistic transformation

Recipient material for biolistic transformation of KB20M1 consisted primarily of conidia. KB20M1 was cultured on CM at 28ºC in the dark for 2 weeks. Conidial masses, and some hyphal fragments, were prepared in sterile water by scraping the culture surface with a bent glass rod and pipetting the resulting slurry to a 15-ml sterile conical tube. Conidial masses were concentrated by centrifugation in a Dynac II Centrifuge (Clay Adams, USA) at 2,300 rpm for 20 min and removing the supernatant with a pipette.Approximately 0.5 cm³ (about 0.2 g) of wet conidial masses were placed in the center of the 9 cm CM plate as the target recipient cells for microprojectile bombardment.

Cosmid pMOcosX (Orbach, 1994) obtained from the Fungal Genetic Stock Center, Kansas City, KS, USA, carrying a hygromycin B resistance gene was used as the exogenous DNA in microprojectile bombardment. The pMOcosX DNA was purified by Aldevron Corp., Fargo, North Dakota, USA.

M5 tungsten microparticles (0.4 µm diameter, Bio-Rad, USA) were surface sterilized in 70% ethanol with vortexing for 3 min, three times. The particles were resuspended at 60 mg/ml in 50% (v/v) glycerol and vortexed at setting 4 on a Fisher Genie 2 Vortex mixer (Fisher Scientific, USA) for 30 min, after which were added 10 µl of pMOcosX DNA at 2 µg/µl, 75 µl of 2.5 M CaCl₂ and 25 µl of 0.1 M Spermidine free base. The suspension was vortexed at the highest speed for 5 min, then incubated on ice for 3 min; this process was repeated three times. The mixture then was incubated on ice for 30 min, centrifuged for 30 sec at 13,000Xg, and the supernatant was removed. The tungsten pellet was gently washed in 300 µl of cold 70% ethanol, and then with 300 µl of cold absolute ethanol. The final cosmid-coated tungsten pellet was briefly vortexed and aliquots of 20 µl containing about 2 µg of DNA and 300 µg of tungsten particles were spread at the center of macrocarriers for a biolistic transformation apparatus (PDS-1000/Helium-driven Biolisticparticle Delivery System, Bio-Rad, USA) and air-dried in a laminar flow hood.

The particle gun was assembled with the distance between the particle launch site and the target cells at 6 cm. A 4.5-cm length of 5 ml pipetter tip was assembled to the macrocarrier's holder to focus the direction of bombardment straight into the target cells (J.P. Fellers, personal communication). The helium gas pressures (rupture disk break pressures) used were 6.2 and 9.3 MPa. The bombardment chamber was evacuated to 85 Kpa prior to bombardment. Five bombardments were conducted at each pressure. Following bombardment, the plates were incubated overnight at room temperature. The bombarded cells were then scraped from the plate surfaces and suspended in 2 ml sterile water. CM agar plates were inoculated with 0.5 ml of the suspension and incubated overnight at room temperature. The bombarded cultures were then overlaid with 12 ml of 0.7% agarose (SeaKem LE) in CM containing 200 µg/ml hygromycin B.

For Southern blot analysis, 15 µg of genomic DNA of putative transformants were isolated and digested singly with XhoI, XbaI, which linearize pMOcosX, and EcoRI. The digested DNA of transformants and undigested DNA of the albino recipient were subjected to gel electrophoresis for 5.5 hrs at 6.25V cm^-1 using 0.8% SeaKem LE agarose in 0.5 X TBE buffer. Random primer labeling was used to incorporate ³²P into 25 ng of pMOcosX used as a probe to detect the integration of this vector in transformants.

These results showed that hygromycin B selection and cosmid pMOcosX can be used in biolistic transformation of whole conidia and mycelial fragments of M. purpureus. Cosmid pMOcosX conferred robust resistance, supporting growth on media containing 1 mg/ml hygromycin B. The two transformants we obtained, both apparently carrying a single copy of the cosmid, did not produce ascospores after eight months growth, compared to abundant ascospore production after 5d growth of the recipient strain. It may be possible to take advantage of this phenomenon with gene expression studies, possibly elucidating the control of ascospore formation in this homothallic fungus. It is not known how frequently this phenomenon occurs in hygromycin B resistant transformants of M. purpureus, but occurred in these two independent transformants, which to our knowledge, are the only transformants of M. purpureus produced to date.

To Dr. Busaba Yongsmith, Faculty of Science, Department of Microbiology, Kasetsart University, Bangkok, Thailand, for providing fungal strain KB20M1.

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