Microbial strains producing sphingoid bases or derivatives thereof
View Patent ↗The present invention provides microbial strains, in particular yeast strains, that produce at least 0.1 mg per g biomass dry weight of a sphingoid base. The present invention further provides a method to obtain sphingoid base-producing microbial strains comprising incubating a population of microbial cells in the presence of a suitable concentration of a toxin, selecting cells that are resistant against said toxin, and isolating cells out of the toxin-resistant cell population that produce at least 0.1 mg per g biomass dry weight of the sphingoid base of Formula I. Optionally, the method further comprises subjecting a population of toxin-resistant microbial cells that produce at least 0.1 mg per g biomass dry weight of the sphingoid base of Formula I to DNA-mediated transformation with a polynucleotide encoding an enzyme of the sphingolipid metabolic pathway. The present invention further provides a polypeptide having dihydroceramide desaturase activity obtainable form Pichia ciferrii.
1. A method for the production of a mixture of sphingoid bases according to Formula I comprising:
fermenting a microbial strain of Pichia ciferrii under conditions conducive to the production of the sphingoid base; and
recovering from the fermentation broth at least 0.1 mg per g biomass dry weight of the mixture of sphingoid bases according to Formula I:
or a salt or ester thereof, wherein:
A-B is CH 2 —CH 2 ; and
R is (CH 2 ) m —X—(CH 2 ) n —CH 3 , where:
m is 0;
n is from 8 to 12; and
X is CH 2 —CH 2 , CH═CH, C≡C, or HC═O—CH 2 ;
wherein the microbial strain of Pichia ciferrii is an isolated syringomycinE-resistant subpopulation of cells that produces at least 0.1 mg per g biomass dry weight of the mixture of sphingoid bases base of Formula I.
2. A method for the production of a mixture of sphingoid bases according to Formula I, comprising:
fermenting a microbial strain of Pichia ciferrii under conditions conducive to the production of the sphingoid base; and
recovering from the fermentation broth at least 10 mg per g biomass dry weight of the mixture of sphingoid bases according to Formula I:
or a salt or ester thereof, wherein:
A-B is CH 2 —CH 2 ; and
R is (CH 2 ) m —X—(CH 2 ) n —CH 3 , where:
m is 0;
n is from 8 to 12; and
X is CH 2 —CH 2 , CH═CH, C≡C, or HC═O—CH 2 ;
wherein the microbial strain of Pichia ciferrii is an isolated toxin-resistant subpopulation of cells that produces at least 10 mg per g biomass dry weight of the mixture of sphingoid bases base of Formula I.
3. A method for producing a mixture of sphingoid bases according to Formula I, comprising:
obtaining a transformed microbial strain of Pichia ciferrii , comprising:
incubating a population of Pichia ciferrii cells in a suitable concentration of a toxin,
selecting a subpopulation of cells that is resistant against the toxin,
isolating cells out of the toxin-resistant subpopulation of cells that produce at least 0.1 mg per g biomass dry weight of the mixture of sphingoid bases of Formula I, and
subjecting the isolated toxin-resistant Pichia ciferrii cells to DNA-mediated transformation with a polynucleotide encoding dihydroceramide desaturase selected from the group consisting of:
a polypeptide with the amino acid sequence according to SEQ ID NO:1, and
a polypeptide with the amino acid sequence having a sequence identity of at least 67% to SEQ ID NO:1;
fermenting the transformed microbial strain under conditions conducive to producing the mixture of sphingoid bases; and
recovering the mixture of sphingoid bases from the fermentation broth;
wherein Formula I is:
or an ester or a salt thereof, where:
A-B is selected from the group consisting of CH 2 —CH 2 and CH═CH; and
R is (CH 2 ) m —X—(CH 2 ) n —CH 3 , where:
m is 0;
n is from 8 to 12; and
X is CH 2 —CH 2 , CH═CH, C≡C, or HC═O—CH 2 .
4. A method for producing a mixture of sphingoid bases according to Formula I, comprising:
obtaining a toxin-resistant, transformed microbial strain of Pichia ciferrii , comprising:
subjecting Pichia ciferrii cells to DNA-mediated transformation with a polynucleotide encoding dihydroceramide desaturase selected from the group consisting of:
a polypeptide with the amino acid sequence according to SEQ ID NO:1, and
a polypeptide with the amino acid sequence having a sequence identity of at least 67% to SEQ ID NO:1,
incubating the transformed population of Pichia ciferrii cells in a suitable concentration of a toxin,
selecting a subpopulation of cells that is resistant against the toxin, and
isolating cells out of the toxin-resistant subpopulation of transformed cells that produce at least 0.1 mg per g biomass dry weight of the mixture of sphingoid bases of Formula I, and
fermenting the isolated toxin-resistant, transformed microbial strain under conditions conducive to producing the mixture of sphingoid bases; and
recovering the mixture of sphingoid bases from the fermentation broth;
wherein Formula I is:
or an ester or a salt thereof, where:
A-B is selected from the group consisting of CH 2 —CH 2 and CH═CH; and
R is (CH 2 ) m —X—(CH 2 ) n —CH 3 , where:
m is 0;
n is from 8 to 12; and
X is CH 2 —CH 2 , CH═CH, C≡C, or HC═O—CH 2 .