Bacteria with improved metabolic capacity
Inventors
Blattner, Frederick R. • Frisch, David A. • Novy, Robert E. • HENKER, Terrance M. • STEFFEN, Eric A. • Sanchez, Jose A. • BLATTNER, Christopher R. • Choi, Hyunsic • Posfai, Gyorgy
Assignees
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Publication Number
US-9902965-B2
Publication Date
2018-02-27
Expiration Date
Abstract
E. coli bacteria comprising genetic modifications to enhance fermentability and production of protein and nucleic acids are provided.
Core Innovation
The invention is a modified Escherichia coli K-12 strain engineered to improve cellular function relevant to recombinant production. The strain is modified to enhance orotate phosphoribosyltransferase activity by introduction of a mutation that complements a native −1 frameshift mutation in the Ribonuclease PH (rph) gene or by deletion of the rph gene. This modification targets pyrE activity through orotate phosphoribosyltransferase activity.
The invention further produces active acetohydroxy acid synthase II by introducing a mutation that complements a native −2 frameshift mutation in the valine-insensitive acetohydroxy synthase II large subunit (ilvG) gene. In the described strain engineering, this complements a frameshift in ilvG and restores a full-length active acetohydroxy acid synthase II, including ilvG_1/ilvG_2 truncation contexts.
In addition, the strain reduces expression of the isocitrate lyase (iclR) and ankyrin repeat protein A (arpA) gene products by deletion of all or part of the iclR and arpA genes. The background and summary rationale described in the provided content frame these changes as part of a reduced-genome/IS-element deletion strategy to improve fed-batch fermentability and recombinant protein and nucleic acid production, including therapeutic proteins (including antibodies) and amplifying heterologous nucleic acids.
Claims Coverage
The provided content contains one independent claim (clm-00001). It defines a modified Escherichia coli K-12 strain with three core genetic engineering features: rph-based enhancement of orotate phosphoribosyltransferase activity, ilvG-based restoration of active acetohydroxy acid synthase II, and deletion-based reduction of iclR and arpA gene product expression.
Enhanced orotate phosphoribosyltransferase activity via rph frameshift complementation or rph deletion
A modified Escherichia coli K-12 strain where the strain is modified to enhance orotate phosphoribosyltransferase activity by introduction of a mutation which complements a native −1 frameshift mutation in the Ribonuclease PH (rph) gene or by deletion of the rph gene.
Active acetohydroxy acid synthase II via ilvG frameshift complementation
A modified Escherichia coli K-12 strain where the strain is modified to produce active acetohydroxy acid synthase II by introduction of a mutation which complements a native −2 frameshift mutation in the valine-insensitive acetohydroxy synthase II large subunit (ilvG) gene.
Reduced iclR and arpA gene products via iclR and arpA deletions
A modified Escherichia coli K-12 strain where the strain is modified to reduce expression of the isocitrate lyase (iclR) and ankyrin repeat protein A (arpA) gene products by deletion of all or part of the iclR and arpA genes.
Across the single independent claim, the inventive coverage centers on three engineered genetic changes in Escherichia coli K-12: rph-based enhancement of orotate phosphoribosyltransferase activity, ilvG-based production of active acetohydroxy acid synthase II, and deletion-based reduction of iclR and arpA gene products expression.
Stated Advantages
Improved fed-batch fermentability.
Increased production of recombinant proteins.
Improved production of nucleic acids (plasmid DNA).
Support of high product formation with high viability during induction in fed-batch fermentation.
Documented Applications
Production of recombinant proteins in fed-batch fermentation, including therapeutic proteins (including antibodies).
Amplifying heterologous nucleic acids (including plasmid DNA).
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