Genetically engineered Vibrio sp. and uses thereof
Inventors
Weinstock, Matthew T • Wilson, Christopher M. • Hesek, Eric D.
Assignees
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Publication Number
US-12331326-B2
Publication Date
2025-06-17
Expiration Date
Abstract
The present disclosure relates to the seminal discovery of a generation and use of genetically engineered Vibrio sp. Provided is the use of the genetically engineered bacteria for the construction, maintenance, manipulation, and/or propagation of DNA constructs; protein expression; protein secretion; vectors and other metabolic tools; metabolic engineering; expression of cellular extracts for cell-free biology; shuttle vectors; cloning vectors; and for synthetic biology applications. The disclosure also relates to the use of the replication machinery of Vibrio sp. as a cloning or expression vector for replication of recombinant DNA constructs. The disclosure also relates to methods of use of the above.
Core Innovation
The document describes genetically engineered Vibrio natriegens organisms configured to support production of a biomolecule using T7 RNA polymerase expression controlled by a recombinant regulatory element. The recombinant nucleic acid includes a nucleotide sequence encoding a T7 RNA polymerase and a regulatory element 5′ to the nucleotide sequence that regulates expression of the T7 RNA polymerase, wherein the regulatory element includes a ribosome binding site having at least 95% sequence identity to SEQ ID NO: 8.
The approach includes introducing a vector into Vibrio natriegens, where the vector comprises a heterologous nucleic acid encoding the biomolecule. The biomolecule is produced by expressing the heterologous nucleic acid in a cell-free extract of the Vibrio natriegens organism, and the biomolecule is isolated.
The disclosure also includes SEQ ID-based components and vector elements related to chromosomal replication machinery associated with chromosome II, enabling use of such replication machinery in cloning, shuttle, and expression vector origins. The disclosure further includes genome engineering approaches and inducible control elements for T7 RNA polymerase, along with weakened ribosome binding sites, competence rendering, and harvesting and isolation of expressed biomolecules.
Claims Coverage
The partial content provides one independent claim, which covers producing a biomolecule using a recombinant Vibrio natriegens cell-free extract system driven by T7 RNA polymerase controlled by a recombinant regulatory element, together with vector-mediated delivery of a heterologous nucleic acid encoding the biomolecule. The dependent claims refine sequence-identity constraints, organism performance constraints, nucleic acid size, biomolecule class, and chromosomal integration and inducible promoter control for the T7 RNA polymerase expression cassette.
T7 RNA polymerase recombinant regulatory element with SEQ ID NO: 8 ribosome binding site identity
The Vibrio natriegens organism comprises a recombinant nucleic acid comprising a nucleotide sequence encoding a T7 RNA polymerase and a recombinant regulatory element 5′ to the nucleotide sequence that regulates expression of the T7 RNA polymerase, wherein the recombinant regulatory element comprises a ribosome binding site having at least 95% sequence identity to SEQ ID NO: 8.
Vector-delivered heterologous nucleic acid encoding the biomolecule
The method contacts a Vibrio natriegens organism with a vector and introduces the vector into the Vibrio natriegens organism, wherein the vector comprises a heterologous nucleic acid encoding the biomolecule.
Cell-free extract expression in Vibrio natriegens to produce the biomolecule and isolation
The method expresses the heterologous nucleic acid in a cell-free extract of the Vibrio natriegens organism, thereby producing the biomolecule, and isolates the biomolecule.
SEQ ID NO: 1-compatible vector replication machinery sequence identity constraint
A vector that contains either the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence with 90% or greater sequence identity to the nucleotide sequence of SEQ ID NO: 1 is used.
Inducible promoter control and chromosomal integration for T7 RNA polymerase
T7 RNA polymerase expression is regulated with an inducible promoter and the inducible promoter and the nucleic acid sequence encoding the T7 RNA polymerase-encoding nucleic acid sequence are integrated into the chromosome of Vibrio natriegens.
Minimum size requirement for heterologous nucleic acid
The heterologous nucleic acid sequence is at least 10 kb in size.
Rapid-growth Vibrio natriegens doubling time threshold
The Vibrio natriegens organism has a doubling time of less than 15 minutes.
Biomolecule limited to protein or peptide
The biomolecule is a protein or peptide.
Overall, the claims coverage is anchored on a recombinant Vibrio natriegens system encoding T7 RNA polymerase under control of a recombinant regulatory element with a ribosome binding site at least 95% identical to SEQ ID NO: 8, combined with vector delivery of a heterologous nucleic acid encoding the biomolecule. The dependent claims further narrow coverage by SEQ ID-based vector sequence-identity constraints, inducible promoter control with chromosomal integration, heterologous nucleic acid size, organism doubling time, and restricting the biomolecule to proteins or peptides.
Stated Advantages
Documented Applications
No documented applications found
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