Reduced genome bacteria with improved genetic stability
Inventors
Blattner, Frederick R. • Csorgo, Balint • Posfai, Gyorgy
Assignees
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Publication Number
US-9914905-B2
Publication Date
2018-03-13
Expiration Date
Abstract
Reduced genome bacteria with improved genetic stability are provided. Also provided are methods of producing polypeptides using the reduced genome bacteria with improved genetic stability.
Core Innovation
A reduced genome Escherichia coli bacterium is engineered to have a genome that is about 5% to about 22% smaller than the genome of its native parent strain. The bacterium lacks all IS1, IS2, IS3, IS4, IS5, IS30, IS150, IS186, IS600, IS911 and IS10 insertion sequences, and the reduction is combined with genetic engineering directed to genetic stability by disabling selected error-prone DNA repair and replication activities.
The bacterium lacks a functional dinB gene, and optionally lacks a functional polB gene and optionally lacks a functional umuDC gene. This engineering is directed to improving genetic stability by using the absence of specified error-prone DNA polymerases in the reduced-genome bacterium. The description links these genomic changes with reduced spontaneous mutation rates and stable genetic maintenance.
The reduced-genome bacterium is described as suitable for high-fidelity nucleic acid maintenance and stable gene expression. It further includes a heterologous nucleic acid encoding a polypeptide operatively linked to an expression control sequence. The engineered bacterium is also described as particularly suitable for maintaining and expressing toxic nucleic acids or proteins that are hard to maintain in standard hosts, with improved stability of a toxic-protein expressing plasmid and lower emergence and accumulation of loss-of-function plasmid mutations.
Claims Coverage
The independent claim defines a genetically engineered reduced-genome Escherichia coli bacterium with a quantified genome-size reduction and a specific set of insertion sequence absences combined with disabling error-prone DNA polymerase genes. Dependent claims further add heterologous nucleic acid content and expression-control linkage, and include an optional method of producing the encoded polypeptide.
Reduced-genome Escherichia coli lacking specified IS elements and error-prone polymerases
A reduced genome Escherichia coli bacterium engineered to have a genome about 5% to about 22% smaller than its native parent strain, lacking all IS1, IS2, IS3, IS4, IS5, IS30, IS150, IS186, IS600, IS911 and IS10 insertion sequences, and lacking a functional dinB gene, optionally lacking a functional polB gene and optionally lacking a functional umuDC gene.
Reduced-genome bacterium lacking a functional umuDC gene
The reduced-genome bacterium of the prior claim, wherein the bacterium lacks a functional umuDC gene.
Reduced-genome bacterium comprising a heterologous nucleic acid
The reduced-genome bacterium of the prior claim comprising a heterologous nucleic acid.
Heterologous nucleic acid encoding a polypeptide operatively linked to an expression control sequence
The reduced-genome bacterium of the prior claim, wherein the heterologous nucleic acid comprises a nucleic acid encoding a polypeptide operatively linked to an expression control sequence.
Producing a polypeptide by expressing and collecting from the engineered bacterium
A method for producing a polypeptide comprising incubating the bacterium under conditions suitable for expressing the polypeptide and collecting the polypeptide.
Overall, the claim set centers on a reduced-genome Escherichia coli bacterium defined by a quantified reduction in genome size, the absence of specified IS insertion sequences, and the loss of functional dinB with optional loss of polB and umuDC, further extending to defined heterologous nucleic acid and expression-control features and an associated polypeptide production method.
Stated Advantages
Improved genetic stability, including reduced spontaneous mutation rates.
High-fidelity nucleic acid maintenance and stable gene expression.
Improved suitability for maintaining and expressing toxic nucleic acids or proteins that are hard to maintain in standard hosts.
Improved stability of a toxic-protein expressing plasmid, with markedly lower emergence and accumulation of loss-of-function plasmid mutations.
Documented Applications
Using the engineered reduced-genome bacterium for stable gene expression and polypeptide production from a heterologous nucleic acid under an expression control sequence.
Maintaining and expressing toxic nucleic acids or proteins that are hard to maintain in standard hosts.
Production of encoded polypeptides by incubating the engineered bacterium under conditions suitable for expressing the polypeptide and collecting the polypeptide.
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