Recombinant Cells and Organisms Having Persistent Nonstandard Amino Acid Dependence and Methods of Making Them

Patent metadata

Field	Value
Publication number	US-2016230176-A1
Application number	US-201415025406-A
Country	US
Kind code	A1
Filing date	Sep 26, 2014
Priority date	Sep 27, 2013
Publication date	Aug 11, 2016
Grant date	—

Abstract

Novel recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making novel recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

First claim

What is claimed is:

1 . A recombinant cell comprising:
An essential polypeptide encoded by an essential nucleic acid sequence, said essential polypeptide having a nonstandard amino acid substitution at a particular position, wherein the absence of the nonstandard amino acid substitution at the particular position disrupts one or any combination of translation, folding and function of the essential polypeptide, and wherein the recombinant cell dies or experiences reduced or no proliferation if one or more of translation, folding or function of the essential polypeptide is disrupted.

2 . The recombinant cell of claim 1 , further comprising one or more standard amino acid substitutions in the essential polypeptide to accommodate the nonstandard amino acid substitution and maintain one or both of proper folding and proper function of the essential polypeptide.

3 . The recombinant cell of claim 2 , wherein the essential polypeptide has been computationally redesigned so that the essential polypeptide can no longer fold properly, function properly or both, when the essential polypeptide expresses a standard amino acid at the particular position.

4 . The essential polypeptide of claim 3 , in which the redesign is performed by random mutagenesis and selection.

5 . The essential polypeptide of claim 3 , in which the redesign is performed by targeted mutagenesis and selection.

6 . The essential polypeptide of claim 3 , in which the redesign is performed computationally using Rosetta software.

7 . The recombinant cell of claim 1 , in which the essential polypeptide is conditionally essential under specific environmental or growth conditions.

8 . The recombinant cell of claim 7 , in which the conditionally essential polypeptide confers antibiotic resistance or is another selection marker.

9 . The recombinant cell of claim 1 , comprising two or more essential polypeptides encoded by two or more essential nucleic acid sequences.

10 . The recombinant cell of claim 1 , wherein the recombinant cell expresses a plurality of nonstandard amino acids.

11 . The recombinant cell of claim 1 , wherein the essential nucleic acid sequence is an essential gene or encodes an essential polypeptide that is essential for survival, growth, or proliferation.

12 . The recombinant cell of claim 1 , wherein the recombinant cell is selected from the group consisting of prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell.

13 . A recombinant cell comprising:
one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the one or more toxins or toxic substances at a particular position, wherein the one or more toxins or toxic substances are activated when the nonstandard amino acid is not present at its particular position, and wherein the activated toxins or toxic substances kill the recombinant cell or prevent or reduce proliferation of the recombinant cell.

14 . The recombinant cell of claim 13 , wherein one or both of folding and function of the one or more toxins or toxic substances are disrupted when the one or more toxins or toxic substances includes the nonstandard amino acid at the particular position.

15 . The recombinant cell of claim 13 , wherein the one or more toxins or toxic substances are selected from the group consisting of one or any combination of barnase, Ccdb, Hok, Fst, ParE, MazF, Kid, ToxN, ReIE, Doc, HipA and Mvpt.

16 . The recombinant cell of claim 13 , wherein multiple copies of a nucleic acid sequence encoding the one or more toxins or toxic substances are present in the cell.

17 . The recombinant cell of claim 13 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell.

18 . A recombinant cell comprising:
an essential polypeptide producing or conferring susceptibility to one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the essential polypeptide at a particular position, wherein the essential polypeptide is activated when the nonstandard amino acid is not present at the particular position, and wherein the activated essential polypeptide confers susceptibility to the one or more toxins or toxic substances, which kills the recombinant cell or prevents or reduces proliferation of the recombinant cell.

19 . The recombinant cell of claim 18 , wherein one or both of folding and function of the essential polypeptide are disrupted when the essential polypeptide includes a nonstandard amino acid at the particular position.

20 . The recombinant cell of claim 18 , wherein the essential polypeptide produces or confers susceptibility to one or more toxins or toxic substances under specific environmental or growth conditions.

21 . The cell of claim 20 , in which the essential polypeptide is selected from the group consisting of one or any combination of SacB Tdk, Ga 1 K, ThyA, To 1 C, TetA, RpsL, and herpes simplex virus thymidine kinase.

22 . The recombinant cell of claim 18 , wherein multiple copies of a nucleic acid sequence encoding the essential polypeptide are present in the recombinant cell.

23 . The recombinant cell of claim 18 , wherein the recombinant cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell.

24 . A recombinant cell comprising:
1) an essential polypeptide encoded by an essential nucleic acid sequence, said essential polypeptide having a nonstandard amino acid substitution, wherein the absence of the nonstandard amino acid substitution disrupts one or any combination of translation, folding and function of the essential polypeptide; and 2) one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the one or more toxins or toxic substances at a particular position, wherein the one or more toxins or toxic substances are activated when the nonstandard amino acid is not present at its particular position, and wherein the activated toxins or toxic substances kill the recombinant cell or prevent or reduce proliferation of the recombinant cell.

25 . The recombinant cell of claim 24 , wherein the cell dies or experiences reduced or no proliferation if one or more of translation, folding or function is disrupted.

26 . The recombinant cell of claim 24 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell.

27 . A recombinant cell comprising:
1) an essential polypeptide encoded by an essential nucleic acid sequence, said essential polypeptide having a nonstandard amino acid substitution, wherein the absence of the nonstandard amino acid substitution disrupts one or any combination of translation, folding and function of the essential polypeptide; and 2) an essential polypeptide producing or c

Assignees

• Harvard College

Inventors

• Church George M
• Mandell Daniel J
• Lajoie Marc J

Classifications

• C12N1/36 Primary

  Adaptation or attenuation of cells · CPC title

• C12N2840/102

  inhibiting translation · CPC title

• C12N15/65 Primary

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title