Methods and molecules for yield improvement involving metabolic engineering
View Patent ↗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.
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.