Preparation of 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof in transgenic organisms

Abstract

The present invention relates to a method for preparing 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof by culturing organisms, in particular yeasts. Furthermore, the invention relates to the preparation of the nucleic acid constructs required for preparing the genetically modified organisms and to said genetically modified organisms, in particular yeasts, themselves.

The invention claimed is: 
     
       1. A method for preparing 7-dehydrocholesterol and/or cholesterol, the method comprising the steps of:
 culturing a yeast organism which, compared to the wild type, has an increased activity of at least one of the activities selected from the group consisting of:
 Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 
 Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8; and 
 Δ24-reductase activity of a Δ24-reductase having the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14; and wherein the yeast organism additionally have increased HMG-CoA-reductase activity of HMG-CoA-reductase having the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24; 
 
 harvesting the yeast after culturing; and 
 isolating the 7-dehydrocholesterol and/or cholesterol after harvesting. 
 
     
     
       2. The method of  claim 1 , wherein the yeast organism additionally has, compared to the wild type, an increased activity of at least one of the activities selected from the group consisting of lanosterol C14-demethylase activity, squalene-epoxidase activity, squalene-synthetase activity and sterol-acyltransferase activity. 
     
     
       3. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. 
     
     
       4. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence of SEQ ID NO: 2. 
     
     
       5. The method of  claim 1 , wherein the yeast organism has an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8. 
     
     
       6. The method of  claim 1 , wherein the yeast organism has an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence of SEQ ID NO: 8. 
     
     
       7. The method of  claim 1 , wherein the yeast organism has an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. 
     
     
       8. The method of  claim 1 , wherein the yeast organism has an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence of SEQ ID NO: 14. 
     
     
       9. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, and an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8. 
     
     
       10. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence of SEQ ID NO: 2, and an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence of SEQ ID NO: 8. 
     
     
       11. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, and an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. 
     
     
       12. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence of SEQ ID NO: 2, and an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence of SEQ ID NO: 14. 
     
     
       13. The method of  claim 1 , wherein the yeast organism has an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8, and an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. 
     
     
       14. The method of  claim 1 , wherein the yeast organism has an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence of SEQ ID NO: 8, and an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence of SEQ ID NO: 14. 
     
     
       15. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8, and an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. 
     
     
       16. The method of  claim 1 , wherein the yeast organism has an increased Δ8-Δ7-isomerase activity of a Δ8-Δ7-isomerase having the amino acid sequence of SEQ ID NO: 2, an increased Δ5-desaturase activity of a Δ5-desaturase having the amino acid sequence of SEQ ID NO: 8, and an increased Δ24-reductase activity of a Δ24-reductase having the amino acid sequence of SEQ ID NO: 14. 
     
     
       17. The method of  claim 1 , wherein the yeast organism additionally has, compared to the wild type, increased HMG-CoA-reductase activity of an HMG-CoA-reductase having the amino acid sequence of SEQ ID NO: 24. 
     
     
       18. The method of  claim 1 , wherein the increased activity is from increased gene expression of a nucleic acid introduced into the yeast organism encoding the Δ8-Δ7-isomerase, Δ5-desaturase, or Δ24-reductase. 
     
     
       19. The method of  claim 3 , wherein the increased Δ8-Δ7-isomerase activity is from increased gene expression of a nucleic acid introduced into the yeast organism encoding the Δ8-Δ7-isomerase. 
     
     
       20. The method of  claim 5 , wherein the increased Δ5-desaturase activity is from increased gene expression of a nucleic acid introduced into the yeast organism encoding the Δ5-desaturase. 
     
     
       21. The method of  claim 7 , wherein the increased Δ24-reductase activity is from increased gene expression of a nucleic acid introduced into the yeast organism encoding the Δ24-reductase.