Sorbitol → L-Leucine

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.

1. additioncontaining 2% polypeptone, 1% yeast
2. extractionextract
3. customthe cells are collected by centrifugation
4. washThe cells are washed with sterilized water (20 ml)
5. waitto stand at 30° C. for about 30 minutes
6. customto give spheroplast
7. customThe spheroplast thus prepared
8. washis washed with 1.2M sorbitol solution three times
9. customThe suspension thus prepared
10. customis divided into a small test tube in a volume of 60 μl
11. additionTo the suspension is added the recombinant plasmid
12. additionAfter mixing well
13. waitto stand at room temperature for 5 to 10 minutes
14. waitto stand at room temperature for about 20 minutes
15. additionThe resulting mixture (each 0.2 ml) is added to a medium (10 ml) [which
16. customis kept at a constant temperature of 45° C
17. additionAfter gently mixing
18. additionthe mixture is added in a layer onto a plate of minimal medium
19. customis previously prepared
20. customis incubated at 30° C.

ORD record

ord-5104867b20ce447cab7d8aab4e5ccc8e

Source dataset

uspto-grants-1991_03

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US04997767