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.

1 . A method for preparing 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof by culturing organisms which, compared to the wild type, have an increased activity of at least one of the activities selected from the group consisting of Δ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductase activity. 
     
     
         2 . The method of  claim 1 , wherein the organisms, compared to the wild type, have an increased activity of at least two of the activities selected from the group consisting of Δ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductase activity. 
     
     
         3 . The method of  claim 1 , wherein the organisms, compared to the wild type, have an increased Δ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductase activity. 
     
     
         4 . The method of  claim 1 , wherein the Δ8-Δ7-isomerase activity is increased by increasing, compared to the wild type, gene expression of a nucleic acid encoding a Δ8-Δ7-isomerase. 
     
     
         5 . The method of  claim 4 , wherein gene expression is increased by introducing into the organism one or more nucleic acids encoding a 48-47-isomerase. 
     
     
         6 . The method of  claim 5 , wherein nucleic acids are introduced, which encode proteins comprising the amino acid sequence SEQ. ID. NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is at least 30% identical at the amino acid level with the sequence SEQ. ID. NO. 2, and having the enzyme property of a Δ8-Δ7-isomerase. 
     
     
         7 . The method of  claim 6 , which comprises introducing a nucleic acid comprising the sequence SEQ. ID. NO. 1. 
     
     
         8 . The method of  claim 1 , wherein the Δ5-desaturase activity is increased by increasing, compared to the wild type, gene expression of a nucleic acid encoding a Δ5-desaturase. 
     
     
         9 . The method of  claim 8 , wherein gene expression is increased by introducing into the organism one or more nucleic acids encoding a 45-desaturase. 
     
     
         10 . The method of  claim 9 , wherein nucleic acids are introduced, which encode proteins comprising the amino acid sequence SEQ. ID. NO. 4 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is at least 30% identical at the amino acid level with the sequence SEQ. ID. NO. 4, and having the enzyme property of a Δ5-desaturase. 
     
     
         11 . The method of  claim 10 , which comprises introducing a nucleic acid comprising the sequence SEQ. ID. NO. 3. 
     
     
         12 . The method of  claim 1 , wherein the Δ24-reductase activity is increased by increasing, compared to the wild type, gene expression of a nucleic acid encoding a Δ24-reductase. 
     
     
         13 . The method of  claim 12 , wherein gene expression is increased by introducing into the organism one or more nucleic acids encoding a Δ24-reductase. 
     
     
         14 . The method of  claim 13 , wherein nucleic acids are introduced, which encode proteins comprising the amino acid sequence SEQ. ID. NO. 6 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is at least 30% identical at the amino acid level with the sequence SEQ. ID. NO. 6, and having the enzymic property of a Δ24-reductase. 
     
     
         15 . The method of  claim 14 , which comprises introducing a nucleic acid comprising the sequence SEQ. ID. NO. 5. 
     
     
         16 . The method of  claim 1 , wherein the organisms, compared to the wild type, additionally have a reduced activity of at least one of the activities selected from the group consisting of C24-methyltransferase activity and Δ22-desaturase activity. 
     
     
         17 . The method of  claim 16 , wherein the organisms, compared to the wild type, have a reduced C24-methyltransferase activity and a reduced 422-desaturase activity. 
     
     
         18 . The method of  claim 16 , wherein the C24-methyltransferase activity is reduced by reducing, compared to the wild type, gene expression of a nucleic acid encoding a C24-methyltransferase. 
     
     
         19 . The method of  claim 18 , wherein an organism is used, which has no functional C24-methyltransferase gene. 
     
     
         20 . The method of  claim 16 , wherein the Δ22-desaturase activity is reduced by reducing, compared to the wild type, gene expression of a nucleic acid encoding a Δ22-desaturase. 
     
     
         21 . The method of  claim 20 , wherein an organism is used, which has no functional Δ22-desaturase gene. 
     
     
         22 . The method of  claim 1 , wherein the organisms additionally have, compared to the wild type, an increased activity of at least one of the activities selected from the group consisting of HMG-CoA-reductase activity, lanosterol C14-demethylase activity, squalene-epoxidase activity, squalene-synthetase activity and sterol-acyltransferase activity. 
     
     
         23 . The method of  claim 22 , wherein the organisms additionally have, compared to the wild type, an increased lanosterol C14-demethylase activity and an increased HMG-CoA-reductase activity. 
     
     
         24 . The method of  claim 22 , wherein the lanosterol C14-demethylase activity is increased by increasing, compared to the wild type, gene expression of a nucleic acid encoding a lanosterol C14-demethylase. 
     
     
         25 . The method of  claim 24 , wherein gene expression is increased by introducing into the organism one or more nucleic acids encoding a lanosterol C14-demethylase. 
     
     
         26 . The method of  claim 25 , wherein nucleic acids are introduced, which encode proteins comprising the amino acid sequence SEQ. ID. NO. 8 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is at least 30% identical at the amino acid level with the sequence SEQ. ID. NO. 8, and having the enzymic property of a lanosterol C14-demethylase. 
     
     
         27 . The method of  claim 26 , which comprises introducing a nucleic acid comprising the sequence SEQ. ID. NO. 7. 
     
     
         28 . The method of  claim 22 , wherein the HMG-CoA-reductase activity is increased by increasing, compared to the wild type, gene expression of a nucleic acid encoding an HMG-CoA reductase. 
     
     
         29 . The method of  claim 28 , wherein gene expression is increased by introducing into the organism a nucleic acid construct comprising a nucleic acid which encodes an HMG-CoA reductase and whose expression in said organism, in comparison with the wild type, is subject to a reduced regulation. 
     
     
         30 . The method of  claim 29 , wherein the nucleic acid construct contains a promoter which, in comparison with the wild-type promoter, is subjected to a reduced regulation in the organism. 
     
     
         31 . The method of  claim 29 , wherein the HMG-CoA reductase-encoding nucleic acid used is a nucleic acid whose expression in the organism, in comparison with the orthologous nucleic acid intrinsic to said organism, is subject to a reduced regulation. 
     
     
         32 . The method of  claim 31 , wherein the HMG-CoA reductase-encoding nucleic acid used is a nucleic acid which encodes the catalytic region of said HMG-CoA reductase. 
     
     
         33 . The method of  claim 32 , wherein nucleic acids are introduced, which encode proteins comprising the amino acid sequence SEQ. ID. NO. 10 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is at least 30% identical at the amino acid level with the sequence SEQ. ID. NO. 10, and having the enzyme property of a HMG-CoA reductase. 
     
     
         34 . The method of  claim 33 , which comprises introducing a nucleic acid comprising the sequence SEQ. ID. NO. 9. 
     
     
         35 . The method of  claim 22 , wherein an organism is used which, compared to the wild type, additionally has an increased squalene-epoxidase activity. 
     
     
         36 . The method of  claim 35 , wherein the squalene-epoxidase activity is increased by increasing, compared to the wild type, gene expression of a nucleic acid encoding a squalene epoxidase. 
     
     
         37 . The method of  claim 36 , wherein gene expression is increased by introducing into the organism one or more nucleic acids encoding a squalene epoxidase. 
     
     
         38 . The method of  claim 37 , wherein nucleic acids are introduced, which encode proteins comprising the amino acid sequence SEQ. ID. NO. 12 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids, which is at least 30% identical at the amino acid level with the sequence SEQ. ID. NO. 12, and having the enzyme property of a squalene epoxidase. 
     
     
         39 . The method of  claim 38 , which comprises introducing a nucleic acid comprising the sequence SEQ. ID. NO. 11. 
     
     
         40 . The method of  claim 1 , wherein the organism used is yeast. 
     
     
         41 . The method of  claim 1 , which comprises harvesting the organism, after culturing, and then isolating 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof from said organism. 
     
     
         42 . A nucleic acid construct, comprising at least one nucleic acid selected from the group consisting of nucleic acids encoding a Δ8-Δ7-isomerase, nucleic acids encoding a Δ5-desaturase and nucleic acids encoding a Δ24-reductase, which are functionally linked with one or more regulatory signals ensuring transcription and translation in organisms. 
     
     
         43 . A nucleic acid construct of  claim 42 , additionally comprising at least one nucleic acid selected from the group consisting of nucleic acids encoding an HMG-CoA reductase, nucleic acids encoding a lanosterol C14-demethylase, nucleic acids encoding a squalene epoxidase, nucleic acids encoding a squalene synthetase and nucleic acids encoding a sterol acyltransferase, which are functionally linked with one or more regulatory signals ensuring transcription and translation in organisms. 
     
     
         44 . A combination of nucleic acid constructs, which comprises at least one nucleic acid construct selected from the groups A to C A nucleic acid construct comprising nucleic acids encoding a Δ8-Δ7-isomerase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms, B nucleic acid construct comprising nucleic acids encoding a Δ5-desaturase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms and C nucleic acid construct comprising nucleic acids encoding a Δ24-reductase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms, and at least one nucleic acid construct selected from the groups D to H D nucleic acid construct comprising nucleic acids encoding an HMG-CoA reductase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms, E nucleic acid construct comprising nucleic acids encoding a lanosterol C14-demethylase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms, F nucleic acid construct comprising nucleic acids encoding a squalene epoxidase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms, G nucleic acid construct comprising nucleic acids encoding a squalene synthetase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms, H nucleic acid construct comprising nucleic acids encoding a sterol acyltransferase, which are functionally linked to one or more regulatory signals ensuring transcription and translation in organisms. 
     
     
         45 . The nucleic acid construct of  claim 42 , wherein the regulatory signals comprise one or more promoters and one or more terminators, which ensure transcription and translation in organisms. 
     
     
         46 . The nucleic acid construct of  claim 42 , wherein regulatory signals ensuring transcription and translation in yeasts are used. 
     
     
         47 . A genetically modified organism, wherein the genetic modification increases at least one of the activities selected from the group consisting of Δ8-Δ7-isomerase activity, Δ5-desaturase activity and Δ24-reductase activity, compared to a wild type. 
     
     
         48 . The genetically modified organism of  claim 47 , wherein the increase of at least one of the activities is caused by an increase in gene expression of at least one nucleic acid selected from the group consisting of nucleic acids encoding a Δ8-Δ7-isomerase, nucleic acids encoding a Δ5-desaturase and nucleic acids encoding a Δ24-reductase, compared to the wild type. 
     
     
         49 . The genetically modified organism of  claim 48 , which contains two or more nucleic acids encoding a Δ8-47-isomerase and/or two or more nucleic acids encoding a Δ5-desaturase and/or two or more nucleic acids encoding a Δ24-reductase. 
     
     
         50 . The genetically modified organism of  claim 47 , wherein the genetic modification additionally reduces at least one of the activities selected from the group consisting of C24-methyltransferase activity and Δ22-desaturase activity compared to a wild type. 
     
     
         51 . The genetically modified organism of  claim 50 , wherein the reduction in at least one of the activities is caused by a reduction in gene expression of at least one nucleic acid selected from the group consisting of nucleic acids encoding a C24-methyltransferase and nucleic acids encoding a Δ 22-desaturase, compared to the wild type. 
     
     
         52 . The genetically modified organism of  claim 51 , which has no functional C24-methyltransferase gene and/or Δ 22-desaturase gene. 
     
     
         53 . The genetically modified organism of  claim 47 , wherein the genetic modification additionally increases at least one of the activities selected from the group consisting of HMG-CoA-reductase activity, lanosterol C14-demethylase activity, squalene-epoxidase activity, squalene-synthetase activity and sterol-acyltransferase activity, compared to a wild type. 
     
     
         54 . The genetically modified organism of  claim 53 , wherein the increase in at least one of the activities is caused by an increase in gene expression of at least one nucleic acid selected from the group consisting of nucleic acids encoding an HMG-CoA-reductase activity, nucleic acids encoding a lanosterol C14-demethylase, nucleic acids encoding a squalene epoxidase, nucleic acids encoding a squalene synthetase and nucleic acids encoding a sterol acyltransferase, compared to the wild type. 
     
     
         55 . The genetically modified organism of  claim 54 , which contains two or more nucleic acids encoding an HMG-CoA reductase and/or two or more nucleic acids encoding a lanosterol C14-demethylase and/or two or more nucleic acids encoding a squalene epoxidase and/or two or more nucleic acids encoding a squalene synthetase and/or two or more nucleic acids encoding a sterol acyltransferase. 
     
     
         56 . The genetically modified organism of  claim 47 , which, compared to the wild type, has an increased content of 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof. 
     
     
         57 . The genetically modified organism of  claim 47 , wherein the organism used is yeast. 
     
     
         58 . The use of a genetically modified organism  claim 47  for preparing 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof. 
     
     
         59 . The combination of nucleic acid constructs of  claim 44 , wherein the regulatory signals comprise one or more promoters and one or more terminators, which ensure transcription and translation in organisms. 
     
     
         60 . The combination of nucleic acid constructs of  claim 44 , wherein regulatory signals ensuring transcription and translation in yeasts are used.