Methods of cloning nucleic acids or producing proteins in a low endotoxin organism
Inventors
Weinstock, Matthew T • Gibson, Daniel G. • Strimling, Daniel
Assignees
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Publication Number
US-11746321-B2
Publication Date
2023-09-05
Expiration Date
Abstract
The invention provides engineered Vibrio sp. organisms that comprise a genetic modification to either or both of the lpxL and/or lpxM genes. The organisms score substantially lower in an in vitro endotoxin assay versus the unmodified or wild type organism. The organisms preserve substantially the growth rate of the corresponding unmodified organisms. The organisms can also have an exogenous nucleic acid cloned in the organism, or an exogenous nucleic acid encoding a protein, polypeptide, or peptide expressed by the organism, and optionally secreted from the organism.
Core Innovation
The invention relates to engineered recombinant Vibrio sp. organisms for low-endotoxin production and cloning. The organisms comprise an exogenous nucleic acid sequence for cloning, or an exogenous nucleic acid sequence encoding a heterologous protein or peptide for production. The genetic modification includes deletion, inactivation, or disruption of the lpxL gene and/or the lpxM gene to substantially reduce endotoxin while maintaining robust growth compared to a corresponding organism not comprising the modification.
A central aspect is combining substantially reduced endotoxin with a growth rate of at least 60% of the corresponding organism when cultivated under the same conditions. The engineered organism has an endotoxin level of less than 50 EU/ml of purified lipopolysaccharide. This enables harvesting the nucleic acid cloned by the organism, or harvesting the protein or peptide produced by the organism, in a way that addresses endotoxin risk and reduces downstream purification burden.
The document also describes that expression from the engineered Vibrio sp. can be maintained while achieving low endotoxin, including minimum production relative to a corresponding organism. In described embodiments, the exogenous nucleic acid or heterologous protein can be produced under culture conditions, including options where the produced protein is secreted. The approach is demonstrated with examples involving Vibrio natriegens and lpxL/lpxM modifications, including retention of growth rate and endotoxin reduction for purified LPS.
Claims Coverage
The provided independent claim defines a method with multiple inventive requirements: (i) recombinant Vibrio sp. carrying an exogenous nucleic acid for cloning or encoding a heterologous protein/peptide, (ii) genetic modification selected from deletion, inactivation, or disruption of lpxL and/or lpxM to substantially reduce endotoxin, (iii) a minimum growth-rate requirement relative to a corresponding organism, (iv) an endotoxin-level limitation for purified lipopolysaccharide, and (v) harvesting the cloned nucleic acid or the produced protein/peptide.
Recombinant Vibrio sp. for cloning or heterologous protein production
A method wherein culturing a recombinant Vibrio sp. organism comprising an exogenous nucleic acid sequence for cloning, or an exogenous nucleic acid sequence encoding a heterologous protein or peptide for production by the organism, followed by harvesting the nucleic acid cloned by the organism or the protein or peptide produced by the organism.
lpxL/lpxM genetic modification for substantially less endotoxin
The recombinant Vibrio sp. organism includes a genetic modification selected from deletion, inactivation, or disruption of the lpxL gene or the lpxM gene, where the organism produces substantially less endotoxin compared to a corresponding organism not comprising the deletion, inactivation, or disruption and cultivated under the same conditions.
Maintained growth rate during low-endotoxin production
The recombinant Vibrio sp. organism exhibits a growth rate of at least 60% of the growth rate of the corresponding organism when cultivated under the same conditions.
Low endotoxin level for purified lipopolysaccharide
The organism has an endotoxin level of less than 50 EU/ml of purified lipopolysaccharide.
The claim coverage centers on using a recombinant Vibrio sp. organism carrying an exogenous nucleic acid for cloning or heterologous protein/peptide production, while genetically modifying lpxL and/or lpxM to substantially reduce endotoxin, maintaining a growth rate of at least 60% of a corresponding organism, and achieving an endotoxin level of less than 50 EU/ml of purified lipopolysaccharide, with harvesting of the cloned nucleic acid or produced protein/peptide.
Stated Advantages
Substantially reduced endotoxin compared to a corresponding organism not comprising the lpxL/lpxM deletion, inactivation, or disruption.
Maintains robust growth, with a growth rate of at least 60% of the growth rate of the corresponding organism under the same conditions.
Provides an endotoxin level of less than 50 EU/ml of purified lipopolysaccharide.
Documented Applications
Endotoxin-sensitive plasmid DNA preparation using a Vibrio natriegens organism with deletions of lpxL or lpxM, where immunogenicity is described as indistinguishable from blank controls.
Production and harvesting of low-endotoxin purified lipopolysaccharide (LPS) from engineered Vibrio natriegens strains having ΔlpxL and/or ΔlpxM modifications, demonstrating markedly reduced purified LPS endotoxin and immune activation.
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