Sorbitol → L-Leucine

Participants

Reaction Steps

The starting yeast is Saccharomyces cerevisiae AH22 [a leu 2 his4 can 1 (Cir+)], which has been deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Japan under Budapest Treaty as "FERM BP-312". The starting yeast is inoculated in YPD medium (100 ml) containing 2% polypeptone, 1% yeast extract and 2% glucose, and the mixture is incubated at 30° C. overnight, and thereafter, the cells are collected by centrifugation. The cells are washed with sterilized water (20 ml) and suspended in a solution (5 ml) of 1.2M sorbitol and 100 μg/ml zymolyase-60,000 (manufactured by Seikagaku Kogyo K. K., Japan), and the suspension is allowed to stand at 30° C. for about 30 minutes to give spheroplast. The spheroplast thus prepared is washed with 1.2M sorbitol solution three times, and then suspended in a solution (0.6 ml) of 2M sorbitol, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5). The suspension thus prepared is divided into a small test tube in a volume of 60 μl. To the suspension is added the recombinant plasmid pONY-S1 (5 μg) as prepared in the above (3). After mixing well, 0.1M CaCl2 (3 μl) is added thereto in a final concentration of 10 mM CaCl2, and the mixture is allowed to stand at room temperature for 5 to 10 minutes. To the resulting mixture is added each 1 ml of 20% polyethylene glycol 4,000, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5), and the mixture is allowed to stand at room temperature for about 20 minutes. The resulting mixture (each 0.2 ml) is added to a medium (10 ml) [which consists of 22% sorbitol, 2% glucose, 0.7% yeast nitrogen base amino acid, 2% YPD, 20 μg/ml histidine, and 3% agar], which is kept at a constant temperature of 45° C. After gently mixing, the mixture is added in a layer onto a plate of minimal medium containing 1.2M sorbitol which is previously prepared and consists of 0.7% yeast nitrogen base amino acid, 2% glucose, 20 μg/ml histidine and 2% agar and is set thereon. The plate is incubated at 30° C. to give colony of a leucine-non-requiring yeast. The colony is incubated in a Burkholder minimal medium supplemented with histidine (20 μg/ml) [cf. Tohe, A. et al., J. Bacteriol., 113, 727-738 (1973)] to give the desired transformed yeast: Saccharomyces cerevisiae YpONY-S1.

Provenance & Evidence