Use of thermophilic nucleases for degrading nucleic acids

Abstract

The present invention relates to the use of a thermophilic nuclease for degrading nucleic acids in vivo and/or in situ, wherein the thermophilic nuclease is heterologous to the host cell and is produced by the host rather than being added exogenously. The present invention further relates to a genetically modified cell which was produced according to the above method. The present invention is particularly beneficial in inactivating the biological activity of recombinant DNA in biomass or biomass-derived products.

What is claimed is: 
     
       1. A genetically modified yeast or fungal cell which comprises a heterologous nucleic acid sequence encoding a thermophilic TaqI or PhoI nuclease gene, wherein the thermophilic nuclease produced by said thermophilic TaqI or PhoI nuclease gene with an optimum temperature of 50° C. or higher and which is latent at the temperature at which the cell has normal growth, wherein the genetically modified cell produces one or more biological compounds or fine chemicals which are not the thermophilic nuclease, said biological compound being selected from the group consisting of: phytoene, lycopene, beta-carotene, alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin, violaxanthin, adonixanthin, vitamin E, retinol, retinal, retinoic acid, retinyl palmitate, and modified forms thereof. 
     
     
       2. The cell of  claim 1 , wherein the nuclease is a DNA-degrading nuclease. 
     
     
       3. The cell of  claim 2 , wherein the DNA-degrading nuclease is TaqI nuclease. 
     
     
       4. The cell of  claim 2 , wherein the DNA-degrading nuclease is PhoI nuclease. 
     
     
       5. The cell of  claim 1 , wherein the yeast or fungal cell is selected from the group consisting of:  Yarrowia, Candida, Hansenula, Saccharomyces, Mortierella, Schizosaccharomyces, Aspergillus, Fusarium, Trichoderma , and  Thraustochytrium.    
     
     
       6. The cell of  claim 5 , wherein the  Yarrowia  species is  Yarrowia lipolytica.    
     
     
       7. The cell of  claim 1 , wherein the gene of the thermophilic nuclease is codon optimized to match the codon usage bias of the yeast or fungal cell. 
     
     
       8. The cell of  claim 3 , wherein the TaqI nuclease comprises a polypeptide having an amino acid sequence of SEQ ID NO: 3. 
     
     
       9. The cell of  claim 4 , wherein the PhoI nuclease comprises a polypeptide having an amino acid sequence of SEQ ID NO: 10. 
     
     
       10. The cell of  claim 1 , wherein the yeast or fungal cell comprises a combination of more than one thermophilic nuclease gene. 
     
     
       11. A method for degrading the nucleic acids of a host cell in vivo and/or in situ, wherein the method comprises:
 a) growing the host cell at a temperature at which the thermophilic nuclease is latent; and 
 b) degrading the nucleic acids of the host cell by changing the temperature in step (a) to a temperature at which the thermophilic nuclease is active, 
 wherein the host cell is a genetically modified yeast or fungal cell of  claim 1 . 
 
     
     
       12. The method of  claim 11 , wherein the nuclease is a DNA-degrading nuclease and the nucleic acids are DNA. 
     
     
       13. The method of  claim 12 , wherein the DNA of the host cell contains recombinant DNA. 
     
     
       14. The method of any of  claim 13 , wherein the temperature in step (a) is optimal for the growth of the host cell. 
     
     
       15. The method of any of  claim 13 , wherein the degradation is conducted at a temperature that is within about ±5° C. of the optimum temperature of said thermophilic nuclease. 
     
     
       16. The method of  claim 15 , wherein the degradation is conducted at a temperature that is optimal for the activity of the thermophilic nuclease. 
     
     
       17. The method of  claim 11 , wherein the degradation is conducted during the pasteurization of the host cell. 
     
     
       18. The method of  claim 11 , wherein the degradation is conducted in a temperature range between 40° C. and 100° C. 
     
     
       19. The method of  claim 12 , wherein the DNA-degrading nuclease is TaqI nuclease. 
     
     
       20. The method of  claim 19 , wherein the degradation is conducted at a temperature ranging between about 60° C. and about 70° C. 
     
     
       21. The method of  claim 19 , wherein the degradation is conducted at 65° C. 
     
     
       22. The method of  claim 12 , wherein the DNA-degrading nuclease is PhoI nuclease. 
     
     
       23. The method of  claim 22 , wherein the degradation is conducted at a temperature ranging between about 70° C. and about 80° C. 
     
     
       24. The method of  claim 23 , wherein the degradation is conducted at 75° C. 
     
     
       25. The method of  claim 11 , wherein the host cell is selected from a group consisting of:  Yarrowia, Candida, Hansenula, Saccharomyces, Mortierella, Schizosaccharomyces, Aspergillus, Fusarium, Trichoderma , and  Thraustochytrium.    
     
     
       26. The method of  claim 25 , wherein the  Yarrowia  species is  Yarrowia lipolytica.    
     
     
       27. The method of  claim 11 , wherein the gene of the thermophilic nuclease is codon optimized to match the codon usage bias of the host cell. 
     
     
       28. The method of  claim 19 , wherein the TaqI nuclease comprises a polypeptide having an amino acid sequence of SEQ ID NO:3. 
     
     
       29. The method of  claim 22 , wherein the PhoI nuclease comprises a polypeptide having an amino acid sequence of SEQ ID NO: 10. 
     
     
       30. The method of  claim 11 , wherein more than one thermophilic nuclease gene is introduced into the host cell. 
     
     
       31. The method of  claim 13 , wherein said recombinant DNA comprises cleavage sites of the thermophilic nuclease. 
     
     
       32. The method of  claim 13 , wherein said recombinant DNA comprises one cleavage site of the thermophilic nuclease every 500 or fewer nucleotides. 
     
     
       33. A process for the production of a biomass product which is free of active nucleic acid molecules, wherein the biomass product is the product of a host cell, wherein the process comprises:
 a) fermenting the host cell at a temperature at which the thermophilic nuclease is latent; 
 b) degrading the nucleic acids of the host cell by changing the temperature in step (a) to a temperature at which the thermophilic nuclease is active; and 
 c) recovering the biomass product; 
 wherein the order for performing steps b) and step c) may be exchanged, and wherein the host cell is a genetically modified yeast or fungal cell of  claim 1 . 
 
     
     
       34. The process of  claim 33 , wherein the nuclease is a DNA-degrading nuclease and the nucleic acids are DNA. 
     
     
       35. The process of  claim 34 , wherein the DNA of the host cell contains recombinant DNA. 
     
     
       36. The process of any of  claims 33 - 35 , wherein the temperature in step (a) is optimal for the growth of the host cell. 
     
     
       37. The process of any of  claims 33 - 35 , wherein the degradation is conducted at a temperature that is within about ±5° C. of the optimum temperature of said thermophilic nuclease.