Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of .

BACKGROUND

Two reference strains have been sequenced from the mushroom , monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 () and a -aminobenzoic acid (PABA)-auxotrophy in AmutBmut () offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker , respectively.

RESULTS

Gene encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new vector pAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pAde8 in single-vector and co-transformations with -selection were similarly high, unlike for plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pAde8 helped in co-transformations of strains with a -selection vector to overcome suicidal effects by transferred . Co-transformation rates of pAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26-42% for various laccase genes and up to 67% with silencing vectors. The bacterial gene can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase.

CONCLUSIONS

and auxotrophic defects in reference strains OK130 and AmutBmut for complementation in transformation are described. pAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene can also be used as an additional selection marker in OK130, making in combination with successive rounds of transformation possible.