# US 10,118,950 B2 · Platforms for cell-free protein synthesis comprising extracts from genomically recoded E. coli strains having genetic knock-out mutations in release factor 1 (RF-1) and endA

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- Number: US 10,118,950 B2
- Document ID: US10118950B2
- Status: Granted
- Kind: B2
- Application number: 14/840,249
- Filed: 2015-08-31
- Published: 2016-03-03
- Granted: 2018-11-06
- Earliest claimed: 2014-08-30
- Assignees: Northwestern University
- Applicants: Northwestern University
- Inventors: Michael Christoper Jewett; Rey William Martin; Seok Hoon Hong; Yong Chan Kwon; Benjamin James Des Soye
- Web page: https://patentgrep.com/patent/US10118950B2

## CPC

- C07K 14/001: {by chemical synthesis}
- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 207/07006: DNA-directed RNA polymerase (2.7.7.6)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The invention relates to genomically recoded organisms, platforms for preparing sequence defined biopolymers in vitro comprising a cellular extract from a genomically recorded organism, and methods for preparing sequence defined biopolymers in vitro are described. In particular, the invention relates to genomically recoded organisms comprising a strain deficient in release factor 1 (RF-1) or a genetic homolog thereof and at least one of at least one additional genetic knock-out mutation, at least one additional upregulated gene product, or both at least one additional knock-out mutation and at least one additional upregulated gene product.

## Claims

### Claim 1

A platform for preparing a sequence defined biopolymer or a protein in vitro, the platform comprising a cellular extract from a genomically recoded E. coli strain comprising:
(i) a genetic knock-out mutation in release factor 1 (RF-1) or a genetic homolog thereof; and
(ii) a genetic knock-out mutation in endA.

### Claim 2 (depends on 1)

The platform of claim 1, wherein the strain is (a) E. coli strain C321.ΔprfA, (b) E. coli strain rec13.ΔprfA, or a derivative of either (a) or (b), further modified to comprise the genetic knock-out mutations as defined in (i) and (ii).

### Claim 3 (depends on 1)

The platform of claim 1, wherein the cellular extract from the strain is capable of preparing a sequence defined biopolymer or a protein in greater yield and/or purity than a strain that does not have the genetic knock-out mutations as defined in (i) and (ii).

### Claim 4 (depends on 1)

The platform of claim 1, wherein the sequence defined biopolymer or the protein comprises a product prepared from the platform that includes at least 5 unnatural amino acids.

### Claim 5 (depends on 1)

The platform of claim 1 further comprising an orthogonal translation system component configured to incorporate unnatural amino acids.

### Claim 6 (depends on 5)

The platform of claim 5, wherein the orthogonal translation system component is expressed from a plasmid present in the genomically recoded strain, expressed from an integration site in the genome of the genetically recoded strain, co-expressed from both a plasmid present in the genomically recoded strain and an integration site in the genome of the genetically recoded strain, expressed in an in vitro transcription and translation reaction, or added exogenously.

### Claim 7 (depends on 1)

The platform of claim 1 further comprising T7 RNA polymerase.

### Claim 8 (depends on 7)

The platform of claim 7, wherein the T7 RNA polymerase is expressed from a plasmid present in the genetically recoded strain or an integration site in the genome of the genetically recoded strain.

### Claim 9 (depends on 1)

The platform of claim 1, wherein the cellular extract from the genomically recoded strain is a component in a reaction mixture.

### Claim 10 (depends on 1)

A method for cell-free protein synthesis of a sequence defined biopolymer or a protein in vitro, the method comprising contacting a RNA template encoding the sequence defined biopolymer with a reaction mixture comprising the platform of claim 1.

### Claim 11 (depends on 10)

The method of claim 10, wherein the cellular extract from the strain of the genomically recoded strain is capable of preparing the sequence defined biopolymer or the protein in greater yield and/or purity than a strain that (i) is not deficient in release factor 1 (RF-1), and (ii) is not deficient in endA.

### Claim 12 (depends on 10)

The method of claim 10, wherein the sequence defined biopolymer or the protein comprises a product prepared from the method that includes at least 5 unnatural amino acids.

### Claim 13 (depends on 10)

The method of claim 10, wherein the sequence defined biopolymer encoded by the RNA template comprises at least 5 unnatural amino acids and wherein a product prepared from the method includes at least 80% of the encoded unnatural amino acids.

### Claim 14 (depends on 10)

The method of claim 10, wherein the sequence defined biopolymer encoded by the RNA template comprises at least 5 unnatural amino acids and wherein at least 80% of a plurality of products prepared from the method include 100% of the encoded unnatural amino acids.

### Claim 15 (depends on 10)

The method of claim 10, wherein at least 80% of a plurality of products prepared from the method are full length.

### Claim 16 (depends on 10)

The method of claim 10, wherein the sequence defined biopolymer or the protein encodes a therapeutic product, a diagnostic product, a biomaterial product, an adhesive product, a biocomposite product, or an agricultural product.

### Claim 17

A platform for preparing a sequence defined biopolymer or a protein in vitro, the platform comprising a cellular extract from a genomically recoded E. colistrain comprising a genetic knock-out mutation in release factor 1 (RF-1) or a genetic homolog thereof and the genomically recoded strain further comprising at least one additional upregulated gene product, wherein the at least one addition upregulated gene product improves energy supply, chaperone levels, translation function, ribosome recycling, or any combination thereof, and the at least one additional upregulated gene product comprises a member selected from the group consisting of ackA, ndk, pykF, cdd, dsbC, dnaK, dnaJ, crpE, tig, groS, groL, infA, infB, fusA, efp, lepA, tufB, hslR, ffr, and any combination thereof.

### Claim 18 (depends on 17)

The platform of claim 17, wherein the strain is (a) E. colistrain C321.ΔprfA, (b) E. coli strain rec13.ΔprfA, or a derivative of either (a) or (b)), further modified to comprise the at least one additional upregulated gene product.

### Claim 19 (depends on 17)

The platform of claim 17, wherein a cellular extract from the strain is capable of preparing a sequence defined biopolymer or a protein in greater yield and/or purity than a strain that (i) is not deficient in release factor 1 (RF-1), and (ii) does not have the at least one additional upregulated gene product.

### Claim 20 (depends on 17)

The platform of claim 17, wherein the at least one additional upregulated gene product improves chaperone levels or translation function.

### Claim 21 (depends on 17)

A method for cell-free protein synthesis of a sequence defined biopolymer or a protein in vitro, the method comprising contacting a RNA template encoding the sequence defined biopolymer with a reaction mixture comprising the platform of claim 17.

### Claim 22

A platform for preparing a sequence defined biopolymer or a protein in vitro, the platform comprising a cellular extract from a genomically recoded E. colistrain comprising:
(i) a genetic knock-out mutation in release factor 1 (RF-1) or a genetic homolog thereof;
(ii) a genetic knock-out mutation in endA; and
(iii) a genetic knock-out mutation in a gene selected from the group consisting of mazF, ma, mb, rne, gor, Ion, ompT, gdhA, gshA, sdaA, sdaB, speA, WaaL, tnaA, glpK, and any combination thereof.

### Claim 23

A platform for preparing a sequence defined biopolymer or a protein in vitro, the platform comprising a cellular extract from a genomically recoded E. colistrain comprising:
(i) a genetic knock-out mutation in release factor 1 (RF-1) or a genetic homolog thereof;
(ii) a genetic knock-out mutation in endA; and
(iii) a genetic knock-out mutation in a gene selected from the group consisting of mazF, rne, gor, and a combination thereof.