Process for preparing genetically transformed yeasts capable of producing a molecule of interest at a high titre

Abstract

The subject of the present invention is a process for preparing a genetically modified yeast by multicopy integration of at least four expression cassettes, allowing the production of a molecule of interest at high titre. The subject of the present invention is also yeasts transformed according to said process, and the use thereof for producing hydrocortisone.

1 . A method for preparing a yeast isolate producing hydrocortisone, comprising:
 (a) providing two integration plasmids with four expression cassettes, each integration plasmid comprising at least two expression cassettes and optionally a selectable marker, wherein the four expression cassettes are P450scc, adrenodoxin (ADX), P450c11, and 36-hydroxysteroid dehydrogenase (3β-HSD)   (b) stably integrating multiple copies of the two integration plasmids into a population of yeast cells, by co-transforming the plasmids into the yeast,   (c) performing a primary screen to select at least 30 yeast clones, wherein selection of the clones is based on the presence of the expression cassettes, or selection for the expression of the selectable marker when such a marker is present, and   (d) performing a functional secondary screen on the at least 30 yeast clones selected in the primary screen, to identify a yeast isolate producing hydrocortisone.   
     
     
         2 . The method of  claim 1 , wherein from 5 to 20 copies of the plasmids are integrated. 
     
     
         3 . The method of  claim 1 , wherein from 8 to 12 copies of the plasmids are integrated. 
     
     
         4 . The method of  claim 1 , wherein at least 40 clones are selected by the primary screen. 
     
     
         5 . The method of  claim 1 , wherein the yeast is  Saccharomyces cerevisae.    
     
     
         6 . The method of  claim 1 , wherein at least one of the plasmids comprises an auxotrophic selectable marker. 
     
     
         7 . The method of  claim 6 , wherein the auxotrophic marker is selected from the group consisting of ADE2, URA3, HIS3, LEU2, TRP1, and LYS2. 
     
     
         8 . The method of  claim 1 , wherein at least one of the plasmids comprises a selectable marker which is a resistance marker. 
     
     
         9 . The method of  claim 8 , wherein the resistance marker is selected from the group consisting of natMX, phMX, and KanMX. 
     
     
         10 . The method of  claim 7 , wherein one of the plasmids comprises URA3 and the other plasmid comprises ADE2. 
     
     
         11 . The method of  claim 10 , wherein the ADE2 gene encodes a truncated, inactive protein. 
     
     
         12 . The method of  claim 1 , wherein the yeast produces at least 100 mg/L hydrocortisone. 
     
     
         13 . The method of  claim 1 , wherein at least 85% of the steroid produced by the yeast is hydrocortisone. 
     
     
         14 - 16 . (canceled) 
     
     
         17 . A method for preparing a genetically transformed yeast producing a high titre of a molecule of interest, comprising:
 (a) providing two integration plasmids with up to four expression cassettes, each integration plasmid comprising at least two expression cassettes and optionally a selectable marker, wherein the different expression cassettes encode proteins which are members of a metabolic pathway involved in the synthesis of the molecule of interest;   (b) stably integrating multiple copies of the two integration plasmids into a population of yeast cells by co-transforming the plasmids into the yeast;   (c) performing a primary screen to select at least 30 yeast clones, wherein selection of the clones is based on the presence of the expression cassettes, or selection for the expression of the selectable marker when such a marker is present; and   (d) performing a functional secondary screen on the at least 30 yeast clones selected in the primary screen, to identify a yeast isolate producing a high titre of a molecule of interest.   
     
     
         18 . The method of  claim 17 , wherein from 5 to 20 copies of the plasmids are integrated. 
     
     
         19 . The method of  claim 17 , wherein from 8 to 12 copies of the plasmids are integrated. 
     
     
         20 . The method of  claim 17 , wherein each expression cassette is selected from the group consisting of an endogenous DNA sequence, an exogenous DNA sequence, and heterogeneous DNA sequences. 
     
     
         21 . The method of  claim 17 , wherein at least 40 clones are selected by the primary screen. 
     
     
         22 . The method of  claim 17 , wherein the yeast is  Saccharomyces cerevisae.    
     
     
         23 . The method of  claim 17 , wherein at least one of the plasmids comprises an auxotrophic selectable marker. 
     
     
         24 . The method of  claim 23 , wherein the auxotrophic marker is selected from the group consisting of ADE2, URA3, HIS3, LEU2, TRP1, and LYS2. 
     
     
         25 . The method of  claim 17 , wherein at least one of the plasmids comprises a selectable marker which is a resistance marker. 
     
     
         26 . The method of  claim 25 , wherein the resistance marker is selected from the group consisting of natMX, phMX, and KanMX. 
     
     
         27 . The method of  claim 24 , wherein one of the plasmids comprises URA3 and the other plasmid comprises ADE2. 
     
     
         28 . The method of  claim 27 , wherein the ADE2 gene encodes a truncated, inactive protein. 
     
     
         29 . The method of  claim 17 , wherein the expression cassettes comprise genes involved in the hydrocortisone biosynthesis pathway. 
     
     
         30 . The method of  claim 29 , wherein the functional secondary screen is an assay to determine the amount of hydrocortisone produced by each of the yeast clones. 
     
     
         31 . The method of  claim 17 , wherein the functional secondary screen is an assay to determine the amount of the molecule of interest produced by each of the yeast clones. 
     
     
         32 - 34 . (canceled)