Methods and molecules for yield improvement involving metabolic engineering

Abstract

The invention features methods and compositions relating to cells that have been engineered to reduce or eliminate proteins having enzymatic activity that interfere with the expression of a metabolic product.

What is claimed is: 
     
       1. A cell comprising:
 (a) an engineered protein expressed from a plasmid, wherein the protein comprises a first moiety having a metabolic enzymatic activity engineered to fuse with a second moiety having a degradation tag,
 wherein the cell does not contain a functional chromosomal copy of the first moiety, 
 wherein the cell comprises a mutation or deletion in the chromosomal copy of the first moiety, 
 
 wherein expression of the engineered protein is under the control of a regulatory system comprising a regulated promoter, and 
 (b) a non-native degradation protein selected from an adaptor, unfoldase, or protease. 
 
     
     
       2. The cell of  claim 1 , wherein said second moiety differs from the amino acid sequence of SEQ ID NO: 1 by at most four amino acid substitutions or deletions. 
     
     
       3. The cell of  claim 2 , wherein said second moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-2 and 4-10. 
     
     
       4. The cell of  claim 1 , wherein said regulated promoter is selected from the group consisting of a lac operon promoter, a nitrogen-regulated promoter, a quorum sensing promoter, and a temperature-sensitive promoter. 
     
     
       5. The cell of  claim 1 , wherein said cell is a microbial cell. 
     
     
       6. The cell of  claim 5 , wherein said cell is a bacterial cell. 
     
     
       7. The cell of  claim 5 , wherein said cell is a fungal cell. 
     
     
       8. A method for producing a desired product, said method comprising:
 (a) culturing in a suitable medium the cell of  claim 1  under conditions that allow expression of the engineered protein; and 
 (b) recovering said desired product from said cell or said medium. 
 
     
     
       9. The cell of  claim 1 , wherein said degradation protein includes one or more of: SspB adaptor protein, ClpX unfoldase, ClpA unfoldase, ClpP protease, and ClpS adaptor. 
     
     
       10. The cell of  claim 9 , wherein
 the SspB adaptor protein comprises the amino acid sequence of SEQ ID NO: 25 or 27; or 
 the ClpX unfoldase comprises the amino acid sequence of SEQ ID NO: 22; or the ClpA unfoldase comprises the amino acid sequence of SED ID NO: 23; or the ClpP protease comprises the amino acid sequence of SEQ ID NO: 24; or the ClpS adaptor comprises the amino acid sequence of SEQ ID NO: 26. 
 
     
     
       11. The cell of  claim 1 , wherein said second moiety comprises the sequence of SEQ ID NO: 3 or 11. 
     
     
       12. The cell of  claim 1 , wherein expression of the engineered protein under the control of the regulatory system is increased by a regulatory factor added to medium. 
     
     
       13. The cell of  claim 1 , wherein expression of the engineered protein under the control of a regulatory system is reduced by reduction of a regulatory factor in medium. 
     
     
       14. The cell of  claim 13 , wherein reduced expression of the engineered protein results in a reduction in the amount of the engineered protein in the cell. 
     
     
       15. The cell of  claim 1 , wherein expression of the engineered protein is under the control of a regulatory system comprising a conditionally-replicated plasmid. 
     
     
       16. A cell comprising:
 an engineered protein expressed from a plasmid, wherein the protein comprises a first moiety having a metabolic enzymatic activity engineered to fuse with a second moiety having a degradation tag,
 wherein the cell does not contain a functional chromosomal copy of the first moiety, 
 wherein the cell comprises a mutation or deletion in the chromosomal copy of the first moiety, 
 wherein expression of the engineered protein is under the control of a regulatory system comprising a regulated promoter, 
 
 wherein said regulated promoter is selected from the group consisting of a lac operon promoter, a nitrogen-regulated promoter, a quorum sensing promoter, and a temperature-sensitive promoter, and
 a heterologous nucleic acid encoding a degradation protein. 
 
 
     
     
       17. The cell of  claim 16 , wherein said second moiety differs from the amino acid sequence of SEQ ID NO: 1 by at most four amino acid substitutions or deletions. 
     
     
       18. The cell of  claim 17 , wherein said second moiety comprises the amino acid sequence of any one of SEQ ID NOs: 1-2 and 4-10. 
     
     
       19. The cell of  claim 16 , wherein said cell is a microbial cell. 
     
     
       20. The cell of  claim 19 , wherein said cell is a bacterial cell. 
     
     
       21. The cell of  claim 19 , wherein said cell is a fungal cell. 
     
     
       22. The cell of  claim 16 , wherein said wherein said degradation protein includes one or more of: SspB adaptor protein, ClpX unfoldase, ClpP protease, and ClpS adaptor. 
     
     
       23. The cell of  claim 22 , wherein
 the SspB adaptor protein comprises the amino acid sequence of SEQ ID NO: 25 or 27; 
 or the ClpX unfoldase comprises the amino acid sequence of SEQ ID NO: 22; or 
 the ClpA unfoldase comprises the amino acid sequence of SED ID NO: 23; or 
 the ClpP protease comprises the amino acid sequence of SEQ ID NO: 24; or 
 the ClpS adaptor comprises the amino acid sequence of SEQ ID NO: 26. 
 
     
     
       24. The cell of  claim 16 , wherein expression of the engineered protein under control of the regulated promoter is increased by a regulatory factor added to medium. 
     
     
       25. The cell of  claim 16 , wherein expression of the engineered protein under control of the regulated promoter is reduced by reduction of a regulatory factor in medium. 
     
     
       26. The cell of  claim 25 , wherein reduced expression of the engineered protein results in a reduction in the amount of the engineered protein in the cell. 
     
     
       27. The cell of  claim 16 , wherein expression of the engineered protein is under the control of a regulatory system comprising a conditionally-replicated plasmid.