US 10,889,841 B2
Oleaginous yeast variant, method for obtaining thereof and use thereof for lipid production
Giuliana Franzosi, Novara (IT); Daniela Cucchetti, Cuggiono (IT); Daniele Bianchi, Arese (IT); Silvia Galafassi, Laveno Mombello (IT); and Concetta Compagno, Spino D'adda (IT)
Assigned to ENI S.p.A., Rome (IT)
Filed by ENI S.p.A., Rome (IT)
Filed on Jan. 30, 2019, as Appl. No. 16/262,554.
Application 16/262,554 is a division of application No. 15/540,121, granted, now 10,301,656, previously published as PCT/IB2015/060031, filed on Dec. 29, 2015.
Claims priority of application No. MI2014A2292 (IT), filed on Dec. 31, 2014.
Prior Publication US 2019/0161778 A1, May 30, 2019
Int. Cl. C12P 7/26 (2006.01); C12P 7/64 (2006.01); C12R 1/645 (2006.01); C12N 1/16 (2006.01)
CPC C12P 7/6463 (2013.01) [C12R 1/645 (2013.01); C12N 1/16 (2013.01); C12P 2203/00 (2013.01)] 7 Claims
1. A method for obtaining an oleaginous yeast variant, comprising:
i. exposing one or more cells of an oleaginous yeast strain to a mutagenic agent in order to generate random mutations in genomic DNA of the cells;
ii. inoculating and cultivating in a culture medium the cells treated with the mutagenic agent, wherein the culture medium comprises a carbon source at a concentration from 20 g/L to 100 g/L, wherein at least one nitrogen source is present; and
iii. separating yeast cells having a lower density from the cell culture;
wherein step (iii) of said method comprises the sub-steps of:
a) collecting a sample from the culture and adding at least one thickening agent in a suitable amount, to obtain a cell suspension;
b) centrifuging the cell suspension for a time of 1 to 5 minutes at an acceleration of 500 g to 1000 g, to obtain a centrifugation supernatant;
c) collecting a higher fraction of the centrifugation supernatant in order to isolate one or more cells characterized by lower density from the rest of the cell culture;
d) inoculating and cultivating the fraction of the supernatant isolated in c) in a culture medium, comprising a carbon source at a concentration from 20 g/L to 100 g/L, wherein at least one nitrogen source is present at a concentration from 3 g/L to 40 g/L;
e) repeating a) to d) at least 2 times, and
f) isolating one or more single mutant colonies,
and wherein the sub-step a) is carried out by adding sorbitol to the culture sample, as a thickening agent, up to a concentration of 1M to 3M.