Enhanced production of recombinant CRM197 in E. coli
AssigneeScarab Genomics, LLC
InventorsCristopher R. Blattner, David Frisch, Robert E. Novy, Terrance M. Henker, Eric A. Steffen, Frederick R. Blattner, Hyunsic Choi, Gyorgy Posfai, Charles F. Landry
Life of this patent
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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.
Claims33
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.
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.
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.
Dependencies are inferred from the claim text.