Enhanced production of recombinant CRM197 in E. coli
Inventors
BLATTNER, Cristopher R. • Frisch, David • Novy, Robert E. • HENKER, Terrance M. • STEFFEN, Eric A. • Blattner, Frederick R. • Choi, Hyunsic • Posfai, Gyorgy • Landry, Charles F.
Assignees
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Publication Number
US-10479820-B2
Publication Date
2019-11-19
Expiration Date
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.
Core Innovation
The document describes improved recombinant CRM197 production using a reduced-genome E. coli host. A reduced-genome E. coli comprises an expression vector encoding a CRM197 protein fused to an ompA, ompF or ytfQ signal sequence that directs transfer of the CRM197 protein to the periplasm, with the nucleotide sequence operably linked to an expression control sequence and expressed under conditions suitable for expression of the recombinant CRM197 protein.
The problem being addressed is the production of recombinant CRM197, with the document focusing on achieving a yield of at least 1 gram per liter of soluble CRM197 from a reduced-genome E. coli host. The narrative connects reduced-genome features with improved recombinant CRM197 performance and reports substantially increased soluble periplasmic CRM197 compared with conventional approaches.
The document also ties performance to specific reduced-genome modifications, including an E. coli K-12 native parent strain comprising a native −2 frameshift mutation in the ilvG gene and a set of deletions relative to E. coli K-12 MG1655, including deletion of the rph gene, correction of the native −2 frameshift mutation in the ilvG gene to restore active acetohydroxy acid synthase II production, and deletion of all or part of the iclR and arpA genes. In addition, the document describes using periplasm targeting via ompA, ompF or ytfQ fusions together with recombinant expression to obtain soluble CRM197 suitable for the described downstream processing.
Claims Coverage
The provided claim set includes three independent claims, each centered on recombinant CRM197 production in a reduced-genome E. coli system, with inventive features defined by periplasm-targeting CRM197 fusion proteins, specific reduced-genome genetic backgrounds relative to K-12, and achieving a yield threshold of soluble CRM197.
Soluble periplasm-targeted CRM197 fusion expression in reduced genome E. coli
Incubating a reduced genome E. coli comprising an expression vector encoding a CRM197 protein fused to an ompA, ompF or ytfQ signal sequence operably linked to an expression control sequence under conditions suitable for expression, whereby a yield of at least 1 gram per liter of soluble CRM197 is obtained.
Reduced genome defined by ilvG frameshift mutation and specified deletions
The native parent E. coli host is an E. coli K-12 strain comprising a native −2 frameshift mutation in the ilvG gene, with reduced genome modifications relative to the native parent including deletion of at least the listed DNA segments, deletion of the rph gene, correction of the native −2 frameshift mutation in ilvG to restore active acetohydroxy acid synthase II production, and deletion of all or part of the iclR and arpA genes.
Reduced genome E. coli host with specified deleted segments relative to K-12 MG1655
The native parent E. coli strain is a K-12 strain, and the reduced genome E. coli host comprises deletion of at least the listed DNA segments relative to the native parent, including reference to deletion of those segments of the E. coli K-12 strain MG1655 or corresponding DNA segments in another K-12 strain.
Periplasm-targeting CRM197 fusion expression in a reduced genome E. coli host defined by the MG1655-based deletions
A reduced genome E. coli host comprising an expression vector encoding a CRM197 protein fused to an ompA, ompF or ytfQ signal sequence operably linked to an expression control sequence, where the native parent is a K-12 strain comprising a native −2 frameshift mutation in the ilvG gene and the reduced genome host comprises the specified deletions and ilvG-related correction including deletion of rph and deletion of all or part of iclR and arpA genes.
Across the independent claims, the coverage is grounded in expressing recombinant CRM197 as an ompA, ompF or ytfQ signal sequence fusion directed to the periplasm in a reduced-genome E. coli background, together with specific deletion-defined host genotypes relative to K-12 and a defined minimum soluble CRM197 yield.
Stated Advantages
A yield of at least 1 gram per liter of soluble CRM197 is obtained.
Reduced-genome features are described as yielding improved recombinant CRM197 production, including increased soluble periplasmic CRM197.
Targeting CRM197 to the periplasm using ompA, ompF or ytfQ signal sequences is associated with soluble CRM197 production.
Documented Applications
Not explicitly described in patent.
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