Reduced genome E. coli lacking toxin-antitoxin genes
Inventors
Blattner, Frederick R. • Novy, Robert E. • Frisch, David A. • Landry, Charles • Choi, Hyunsic • STEFFEN, Eric A. • Brandon, John
Assignees
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Publication Number
US-11970725-B2
Publication Date
2024-04-30
Expiration Date
Abstract
Provided herein are E. coli host strains with improved capacity for producing recombinant proteins.
Core Innovation
The invention relates to a non-naturally occurring E. coli bacterium having a genome between 4.41 Mb and 2.78 Mb. The bacterium lacks at least a specified set of toxinantitoxin (TA) genes, including yafQ, dinJ, hha, tomB, gnsA, ymcE, yoeB, yefM, mazF, mazE, mazG, cptA/ygfX, and cptB/sdhE.
The invention further specifies that the bacterium lacks additional toxinantitoxin genes such as mqsR, mqsA, higB, higA, yhaV, prlF, ldrD, rdlD, istR-2, tisB, chpB, chpS, ratA, ratB, ldrA, rdlA, ldrB, rdlB, ldrC, rdlC, hokB, sokB, sibA, ibsA, sibB, ibsB, ohsC, shoB, sibC, ibsC, sibD, sibE, and dinQ.
The invention also defines absence of further toxinantitoxin genes including agrA, agrB, ghoT, ghoS, yfeC, yfeD, fic, yhfG, yhjJ, yhjM, yhjN, yjjJ, and ecnA and ecnB, thereby providing a reduced-genome, engineered E. coli K-12/B background. The described approach is presented as enabling long-term continuous fermentation and improved recovery of difficult proteins, including improved release/extraction yields and simplified purification through release of periplasmic proteins into culture medium.
Claims Coverage
The independent claim is defined by the presence of a reduced-genome E. coli with a specific genome-size range and a large specified set of absent toxinantitoxin genes. Dependent claims refine the genotype by adding further deletions, removing specified insertion sequences, constraining the parent strain background, and in at least one dependent introducing a recombinant-polypeptide expression construct.
Reduced-genome E. coli with defined genome-size range
A non-naturally occurring E. coli bacterium having a genome between 4.41 Mb and 2.78 Mb.
Absence of specified toxinantitoxin gene set
The bacterium lacks at least the following toxinantitoxin genes: yafQ, dinJ, hha, tomB, gnsA, ymcE, yoeB, yefM, mazF, mazE, mazG, cptA/ygfX, cptB/sdhE, mqsR, mqsA, higB, higA, yhaV, prlF, ldrD, rdlD, istR-2, tisB, chpB, chpS, ratA, ratB, ldrA, rdlA, ldrB, rdlB, ldrC, rdlC, hokB, sokB, sibA, ibsA, sibB, ibsB, ohsC, shoB, sibC, ibsC, sibD, sibE, dinQ, agrA, agrB, ghoT, ghoS, yfeC, yfeD, fic, yhfG, yhjJ, yhjM, yhjN, yjjJ, ecnA, and ecnB.
Defined parent strain background (K-12 or B)
The parent strain of the bacterium is either a K-12 strain or a B strain.
Optional rnc gene deletion
The bacterium includes a full or partial deletion of the rnc gene.
Absence of specified insertion sequences
The bacterium is further defined as lacking the insertion sequences IS1, IS2, IS3, IS5, IS150, and IS186.
Engineered modifications to alter enzyme activity and delete defined genes
The bacterium is further engineered with specific gene deletions and sequence corrections to alter enzyme activity and restore acetohydroxy acid synthase II production, and to delete all or part of certain genes, including modifications involving rph and native-2 frameshift mutation and deletions affecting ilvG, iclR, and arpA.
Recombinant-polypeptide expression construct using operably linked expression control sequence
The claim family includes a dependent claim that introduces a recombinant-polypeptide expression construct via nucleic acid with an operably linked expression control sequence.
Overall, claim coverage is anchored in a reduced-genome, non-naturally occurring E. coli with a specified genome-size range and absence of an enumerated set of toxinantitoxin genes, with dependent claims further narrowing by optional rnc deletion, removal of specified insertion sequences, selection of K-12/B parent background, additional engineering, and a recombinant-polypeptide expression construct.
Stated Advantages
Enables long-term continuous fermentation.
Improved recovery of difficult proteins.
Increased release/extraction yields of periplasmic proteins into culture medium.
Simplified purification.
Reduced sensitivity to stress-induced loss of production in continuous manufacturing.
Enhanced periplasmic yields after TA gene deletions.
Minimal contamination due to intact-cell harvesting [procedural detail omitted for safety].
Documented Applications
Continuous manufacturing of proteins, including long-term continuous fermentation for producing difficult proteins with improved recovery.
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