# US 10,479,820 B2 · Enhanced production of recombinant CRM197 in E. coli

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- Number: US 10,479,820 B2
- Document ID: US10479820B2
- Status: Granted
- Kind: B2
- Application number: 15/122,891
- Filed: 2015-03-02
- Published: 2017-03-16
- Granted: 2019-11-19
- Earliest claimed: 2014-03-03
- Assignees: Scarab Genomics, LLC
- Applicants: Scarab Genomics, LLC
- Inventors: Cristopher R. Blattner; David Frisch; Robert E. Novy; Terrance M. Henker; Eric A. Steffen; Frederick R. Blattner; Hyunsic Choi; Gyorgy Posfai; Charles F. Landry
- Web page: https://patentgrep.com/patent/US10479820B2

## CPC

- C07K 14/34: from Corynebacterium (G)
- C12N 1/08: Reducing the nucleic acid content
- C12N 1/20: Bacteria; Culture media therefor
- 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)

## Abstract

Rediced genome or native K12 strain E. coli bacteria comprising expression vectors encoding a recombinant CRM197 protein and their use in the production of CRM 197 is provided. The CRM 197 protein may be fused to a signal sequence that directs the expressed CRM197 protein to the periplasm of the E. coli host.

## Claims

### Claim 1

A method for producing a recombinant CRM197 in a reduced genome E. coli host, the method comprising incubating a reduced genome E. coli comprising an expression vector comprising a nucleotide sequence encoding a CRM197 protein fused to an ompA, ompF or ytfQ signal sequence that directs transfer of the CRM197 protein to the periplasm, said nucleotide sequence operably linked to an expression control sequence, under conditions suitable for the expression of the recombinant CRM197 protein, whereby a yield of at least 1 gram per liter of soluble CRM197 is obtained, wherein the native parent strain of the reduced genome E. coli host is an E. coli K-12 strain comprising a native −2 frameshift mutation in the ilvG gene, and wherein the reduced genome E. coli host comprises the following modifications relative to the native parent E. coli strain: (i) deletion of at least the following DNA segments: b0245-b0301, b0303-b0310, b1336-b1411, b4426-b4427, b2441-b2450, b2622-b2654, b2657-b2660, b4462, b1994-b2008, b4435, b3322-b3338, b2349-b2363, b1539-b1579, b4269-b4320, b2968-b2972, b2975-b2977, b2979-b2987, b4466-4468, b1137-b1172, b0537-b0565, b0016-b0022, b4412-b4413, b0577-b0582, b4415, b2389-b2390, b2392-b2395, b0358-b0368, b0370-b0380, b2856-b2863, b3042-b3048, b0656, b1325-b1333, b2030-b2062, b2190-b2192, b3215-b3219, b3504-b3505, b1070-b1083, b1878-b1894, b1917-b1950, b4324-b4342, b4345-b4358, b4486, b0497-b0502, b0700-b0706, b1456-b1462, b3481-b3484, b3592-b3596, b0981-b0988, b1021-b1029, b2080-b2096, b4438, b3440-b3445, b4451, b3556-b3558, b4455, b1786, b0150-b0153 and b2945 of the E. coli K12 strain MG1655 or the corresponding DNA segments in another E. coli K12 strain (ii) deletion of the rph gene to enhance orotate phosphoribosyltransferase activity (iii) correction of the native −2 frameshift mutation in the ilvG gene in order to restore the active acetohydroxy acid synthase II production and (iv) deletion of all or part of the iclR and arpA genes.

### Claim 2 (depends on 1)

The method of claim 1, whereby a yield of at least 2 grams per liter of soluble CRM197 is obtained.

### Claim 3 (depends on 1)

The method of claim 1, wherein the signal sequence is OmpF or YtfQ.

### Claim 4 (depends on 3)

The method of claim 3, wherein the signal sequence is YtfQ.

### Claim 5 (depends on 1)

The method of claim 1, wherein the reduced genome E. coli has additionally deleted therefrom at least the following DNA segments: b0315-b0331, b0333-b0341, b0346-b0354, b2481-b2492, b2219-b2230, b4500, b3707-b3723, b0644-b0650, b4079-4090, b4487, b4092-b4106, b0730-b0732, b3572-b3587, b1653, b2735-b2740, b2405-b2407, b3896-b3900, b1202, b4263-b4268, b0611, b2364-b2366, b0839, b0488-b0500, and b0502 of the E. coli K-12 strain MG1655 or the corresponding DNA segments in another E. coli K-12 strain.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reduced genome E. coli comprises a functional recA (b2699) gene.

### Claim 7 (depends on 1)

The method of claim 1, wherein the reduced genome E. coli comprises a relA gene having at least one point mutation at position 547 or 548 of the coding sequence of the relA gene, wherein the mutation is selected from one or more of: a G→A mutation at position 547, a G→T mutation at position 547, a C→G mutation at position 548, or a C→T mutation at position 548.

### Claim 8 (depends on 1)

The method of claim 1, wherein the CRM197 nucleotide sequence is optimized for expression in the E. coli host cell.

### Claim 9 (depends on 1)

The method of claim 1 comprising
(a) growing the reduced genome E. coli and
(b) inducing expression of CRM197.

### Claim 10 (depends on 9)

The method of claim 9, wherein the method is carried out in a fermentor.

### Claim 11 (depends on 9)

The method of claim 9, wherein step (a) comprises growing the reduced genome E. coli at 37° C. for up to 19 hours followed by growth at about 20-30° C. prior to and subsequent to step (b).

### Claim 12 (depends on 9)

The method of claim 9, wherein steps (a) and (b) are performed in growth medium that does not comprise serum, yeast extract or other animal by-products.

### Claim 13 (depends on 10)

The method of claim 10, wherein in step (a) the pH ranges between 6.5 and 7.5.

### Claim 14 (depends on 13)

The method of claim 13, wherein the pH is maintained using a phosphate buffer, a Tris buffer or a histidine buffer.

### Claim 15 (depends on 14)

The method of claim 14, wherein the buffer is a phosphate buffer.

### Claim 16 (depends on 9)

The method of claim 9, wherein expression is induced in step (b) by addition of a suitable amount of IPTG.

### Claim 17 (depends on 10)

The method of claim 10, wherein expression is induced at an OD600 of from 100 to 400.

### Claim 18 (depends on 10)

The method of claim 10 wherein the fermentor contains 0.5-50,000 liters of culture.

### Claim 19 (depends on 9)

The method of claim 9 comprising a further step of (c) mechanically disrupting the cultured reduced genome E. coli cells in the absence of detergent and centrifuging the resulting cell lysate to obtain a soluble fraction.

### Claim 20 (depends on 19)

The method of claim 19, wherein the mechanical disruption comprises sonication or microfluidization.

### Claim 21 (depends on 19)

The method of claim 19, wherein CRM197 is purified from the soluble fraction by one or more purification steps.

### Claim 22 (depends on 21)

The method of claim 21, wherein the one or more purification steps comprises hydrophobic interaction chromatography and/or anion exchange chromatography.

### Claim 23 (depends on 22)

The method of claim 22, wherein the purification steps comprise hydrophobic interaction chromatography followed by anion exchange chromatography.

### Claim 24 (depends on 1)

The method of claim 1, wherein the native parent E. coli strain is K-12 strain MG1655.

### Claim 25 (depends on 1)

The method of claim 1, wherein the reduced genome E. coli lacks a functional recA (b2699) gene.

### Claim 26 (depends on 1)

The method of claim 1, wherein the reduced genome E. coli does not comprise insertion sequences.

### Claim 27 (depends on 1)

The method of claim 1, wherein the reduced genome E. coli lacks a functional dinB (b0231) gene and optionally lacks functional polB (b0060) and/or umuDC (b1183-b1184) genes.

### Claim 28

A reduced genome E. coli host comprising an expression vector comprising a nucleotide sequence encoding a CRM197 protein fused to an ompA, ompF or ytfQ signal sequence that directs transfer of the CRM197 protein to the periplasm, wherein said nucleotide sequence is operably linked to an expression control sequence, wherein the native parent E. coli strain of the reduced genome E. coli host is a K-12 strain comprising a native−-2 frameshift mutation in the ilvG gene, and wherein the reduced genome E. coli host comprises the following modifications relative to the native parent E. coli strain: (i) deletion of at least the following DNA segments: b0245-b0301, b0303-b0310, b1336-b1411, b4426-b4427, b2441-b2450, b2622-b2654, b2657-b2660, b4462, b1994-b2008, b4435, b3322-b3338, b2349-b2363, b1539-b1579, b4269-b4320, b2968-b2972, b2975-b2977, b2979-b2987, b4466-4468, b1137-b1172, b0537-b0565, b0016-b0022, b4412-b4413, b0577-b0582, b4415, b2389-b2390, b2392-b2395, b0358-b0368, b0370-b0380, b2856-b2863, b3042-b3048, b0656, b1325-b1333, b2030-b2062, b2190-b2192, b3215-b3219, b3504-b3505, b1070-b1083, b1878-b1894, b1917-b1950, b4324-b4342, b4345-b4358, b4486, b0497-b0502, b0700-b0706, b1456-b1462, b3481-b3484, b3592-b3596, b0981-b0988, b1021-b1029, b2080-b2096, b4438, b3440-b3445, b4451, b3556-b3558, b4455, b1786, b0150-b0153 and b2945 of the E. coli K12 strain MG1655 or the corresponding DNA segments in another E. coli K12 strain (ii) deletion of the rph gene to enhance orotate phosphoribosyltransferase activity (iii) correction of the native −2 frameshift mutation in the ilvG gene in order to restore the active acetohydroxy acid synthase II production and (iv) deletion of all or part of the ic1R and arpA genes.

### Claim 29 (depends on 28)

The reduced genome E. coli host according to claim 28, wherein the native parent E. coli strain is K-12 strain MG1655.

### Claim 30

A method for producing a recombinant CRM197 in a reduced genome E. coli host comprising incubating a reduced genome E. coli comprising an expression vector comprising a nucleotide sequence encoding a CRM197 protein fused to an ompA, ompF or ytfQ signal sequence that directs transfer of the CRM197 protein to the periplasm, said nucleotide sequence operably linked to an expression control sequence, under conditions suitable for the expression of the recombinant CRM197 protein, whereby a yield of at least 1 gram per liter of soluble CRM197 is obtained, wherein the native parent E. coli strain of the reduced genome E. coli host is a K-12 strain and wherein the reduced genome E. coli host comprises a deletion of at least the following DNA segments relative to the native parent E. coli strain: b0245-b0301, b0303-b0310, b1336-b1411, b4426-b4427, b2441-b2450, b2622-b2654, b2657-b2660, b4462, b1994-b2008, b4435, b3322-b3338, b2349-b2363, b1539-b1579, b4269-b4320, b2968-b2972, b2975-b2977, b2979-b2987, b4466-4468, b1137-b1172, b0537-b0565, b0016-b0022, b4412-b4413, b0577-b0582, b4415, b2389-b2390, b2392-b2395, b0358b0368, b0370-b0380, b2856-b2863, b3042-b3048, b0656, b1325-b1333, b2030-b2062, b2190-b2192, b3215-b3219, b3504-b3505, b1070-b1083, b1878-b1894, b1917-b1950, b4324-b4342, b4345-b4358, b4486, b0497-b0502, b0700-b0706, b1456-b1462, b3481-b3484, b3592-b3596, b0981-b0988, b1021-b1029, b2080-b2096, b4438, b3440-b3445, b4451, b3556-b3558, b4455, b1786, b0150-b0153 and b2945 of the E. coli K-12 strain MG1655 or the corresponding DNA segments in another E. coli K-12 strain.

### Claim 31 (depends on 30)

The method of claim 30, wherein the reduced genome E. coli host is strain MDS42.

### Claim 32 (depends on 30)

The method of claim 30, wherein the reduced genome E. coli has additionally deleted therefrom at least the following DNA segments: b0315-b0331, b0333-b0341, b0346-b0354, b2481-b2492, b2219-b2230, b4500, b3707-b3723, b0644-b0650, b4079-4090, b4487, b4092-b4106, b0730-b0732, b3572-b3587, b1653, b2735-b2740, b2405-b2407, b3896-b3900, b1202, b4263-b4268, b0611, b2364-b2366, b0839, b0488-b0500, and b0502 of the E. coli K-12 strain MG1655 or the corresponding DNA segments in another E. coli K-12 strain.

### Claim 33 (depends on 10)

The method according to claim 10, wherein the method comprises fed-batch fermentation.