Synthetic gene clusters

Patent metadata

Field	Value
Publication number	US-9957509-B2
Application number	US-201615288916-A
Country	US
Kind code	B2
Filing date	Oct 7, 2016
Priority date	Jun 16, 2011
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

Methods for making synthetic gene clusters are described.

First claim

What is claimed is:

1. A bacterial nitrogen reduction expression system comprising nucleic acids encoding:
at least one operon comprising a plurality of coding sequences for a set of polypeptides encoded by genes collectively associated with nitrogen fixation within a cell, wherein at least one of the plurality of coding sequences comprises at least one non-native codon;
a heterologous promoter region that directs expression of the at least one operon; and
a heterologous transcriptional controller coding sequence that encodes a protein that directs expression of the at least one operon of the expression system, wherein the protein binds directly or indirectly to the heterologous promoter region.

2. The bacterial nitrogen reduction expression system of claim 1 , wherein the genes collectively associated with nitrogen fixation within a cell are selected from the group consisting of: nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifB, and nifQ.

3. The bacterial nitrogen reduction expression system of claim 1 , wherein the heterologous promoter of the one or more of the coding sequences causes the one or more coding sequences to be expressed at an expression level which is associated with maximal nitrogenase activity.

4. The bacterial nitrogen reduction expression system of claim 1 , wherein the heterologous promoter region is a promoter which does not natively regulate a gene associated with nitrogen fixation.

5. A bacterial nitrogen reduction expression system comprising nucleic acids encoding:
at least one operon comprising a plurality of coding sequences for a set of polypeptides encoded by genes collectively associated with nitrogen fixation within a cell, wherein at least one of the plurality of coding sequences comprises at least one non-native codon;
a heterologous promoter region that directs expression of the at least one operon, wherein the heterologous promoter region is from the same species as the genes collectively associated with nitrogen fixation; and
a transcriptional controller coding sequence that encodes a protein that directs expression of the at least one operon of the expression system, wherein the protein binds directly or indirectly to the heterologous promoter region, and wherein the transcriptional controller does not regulate the genes collectively associated with nitrogen fixation under native regulation.

6. The bacterial nitrogen reduction expression system of claim 5 , wherein the genes collectively associated with nitrogen fixation within a cell are selected from the group consisting of: nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifB, and nifQ.

7. The bacterial nitrogen reduction expression system of claim 5 , wherein the coding sequences have been modified to reduce a predicted RNA secondary structure.

8. The bacterial nitrogen reduction expression system of claim 5 , wherein the heterologous promoter of the at least one operon causes a coding sequence of the operon to be expressed at an expression level which is associated with maximal nitrogenase activity.

9. The bacterial nitrogen reduction expression system of claim 5 , wherein the heterologous promoter of the operon causes two or more coding sequences to be expressed at an expression level which causes maximal nitrogenase activity.

10. The bacterial nitrogen reduction expression system of claim 5 , wherein the heterologous promoter causes each coding sequence of the operon to be expressed at an expression level which causes maximal nitrogenase activity.

11. The bacterial nitrogen reduction expression system of claim 5 , wherein the heterologous promoter causes each coding sequence of the operon to be expressed at an expression level which is associated with maximal nitrogenase activity.

12. A bacterial nitrogen reduction expression system comprising nucleic acids encoding:
at least one operon comprising a plurality of coding sequences for a set of polypeptides encoded by genes collectively associated with nitrogen fixation within a cell, wherein at least one of the plurality of regulatory coding sequences has been synonymously mutated to remove internal regulation;
a genetically engineered promoter region that directs expression of the at least one operon; and
a transcriptional controller coding sequence that encodes a protein that directs expression of the at least one operon of the expression system, wherein the protein binds directly or indirectly to the heterologous promoter region, and wherein the transcriptional controller does not regulate the genes collectively associated with nitrogen fixation under native regulation.

13. The bacterial nitrogen reduction expression system of claim 12 , wherein the coding sequences have been modified to reduce a predicted RNA secondary structure.

14. The bacterial nitrogen reduction expression system of claim 12 , wherein the genes collectively associated with nitrogen fixation within a cell are selected from the group consisting of: nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifB, and nifQ.

15. The bacterial nitrogen reduction expression system of claim 12 , wherein the heterologous promoter of the at least one operon causes a coding sequence of the operon to be expressed at an expression level which is associated with maximal nitrogenase activity.

16. The bacterial nitrogen reduction expression system of claim 12 , wherein the heterologous promoter of the operon causes two or more coding sequences to be expressed at an expression level which is associated with maximal nitrogenase activity.

Assignees

• Univ California

Inventors

• Mirsky Ethan
• Temme Karsten
• Voigt Christopher A
• Zhao Dehua

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• C12Y118/06001

  Nitrogenase (1.18.6.1) · CPC title

• C12N9/0095

  acting on iron-sulfur proteins as donor (1.18) · CPC title

• C12N15/635 Primary

  Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline · CPC title