Recombinant strains and medium formulation for enhancing secretion titer using a type III secretion system
Inventors
Ercek, Danielle Tullman • Burdette, Lisa A. • Wong, Han Teng
Assignees
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Publication Number
US-12565519-B2
Publication Date
2026-03-03
Expiration Date
Abstract
The present disclosure provides a recombinant Salmonella strain having a Type III secretion system (T3SS) comprising mutation which enhance protein expression and production. Additionally, methods and kits for using the recombinant Salmonella strain for producing a protein of interest are provided. Additionally, an optimized medium that increases protein expression in a Salmonella strain having a Type III secretion system (T3SS) is provided.
Core Innovation
The invention relates to recombinant Salmonella strains engineered to improve heterologous protein production by leveraging a Type III secretion system (T3SS). A recombinant Salmonella strain is described as having a T3SS and genetic modifications that increase hilD expression and enhance protein secretion into an extracellular medium.
In particular, the strain includes an insertion of a protein coding sequence between the hil coding sequence and the hilD 3′UTR suitable to increase hilD expression, and the strain includes deletion of hilE. The protein coding sequence encodes a β-barrel protein, and the document describes how strain engineering changes in hil regulatory elements are used to increase secretion titer.
The document further describes producing heterologous proteins using T3SS-directed secretion, including use of a targeting polypeptide/secretion tag for secretion into the surrounding medium, and optional cleavable linkers/protease cleavage for processing. The disclosed approach includes use of secretion fractions and downstream purification, and it supports safer recombinant strains through deletions of virulence/pathogenicity genes and pathogenicity islands.
Claims Coverage
The independent claim defines a recombinant Salmonella T3SS strain with specific genetic modifications to increase hilD expression and enhance secretion, including deletion of hilE, and a β-barrel protein coding insertion. Dependent claims further specify T3SS secretion production using a targeting polypeptide, quantitative performance and virulence-reducing deletions, and defined culture medium formats for increased protein secretion.
Insertion between hil coding sequence and hilD 3′UTR for increased hilD expression
A recombinant Salmonella strain having a Type III secretion system (T3SS) with an insertion of a protein coding sequence between the hil coding sequence and the hilD 3′UTR suitable to increase hilD expression in the strain.
Deletion of hilE
A recombinant Salmonella strain having a Type III secretion system (T3SS) wherein the strain includes a deletion of hilE.
β-barrel protein coding insertion
The protein coding sequence inserted between the hil coding sequence and the hilD 3′UTR encodes a β-barrel protein.
T3SS-directed secretion of a heterologous protein via a targeting polypeptide
An in vitro method that expresses a heterologous protein of interest in the recombinant Salmonella strain, where the protein is fused to a targeting polypeptide that directs it to the Type III secretion system (T3SS) and results in secretion of the protein into the surrounding medium.
Increased secretion titer relative to a non-recombinant strain
The recombinant Salmonella strain is characterized by at least a 4-fold higher secretion titer of a protein of interest via the Type III secretion system compared with a non-recombinant Salmonella strain that has a Type III secretion system.
Virulence or pathogenicity reduction by deletion of selected genes and islands
A recombinant Salmonella strain that includes additional modifications to reduce pathogenicity or virulence by deleting at least one of sipB, sipC, sipD, flhDC, pathogenicity island 4 (SPI-4), and pathogenicity island 5 (SPI-5).
Defined medium composition for enhanced protein secretion
A method specifying culturing a recombinant Salmonella strain in a defined medium containing peptone, yeast extract, glycerol or glucose, and potassium phosphate at pH 7.4, with optionally NaCl to increase protein secretion.
Overall, the claim set centers on engineering a recombinant Salmonella T3SS strain by inserting a β-barrel protein coding sequence between the hil coding sequence and the hilD 3′UTR to increase hilD expression and by deleting hilE, together with dependent claim features for T3SS-directed secretion of a heterologous protein, increased secretion titer, optional virulence-reducing deletions, and defined medium composition for enhanced secretion.
Stated Advantages
Increased hilD expression in the strain.
Increased protein secretion titer via the Type III secretion system.
At least a 4-fold higher secretion titer compared with a non-recombinant Salmonella strain.
Reduced pathogenicity or virulence through deletions of virulence/pathogenicity genes and pathogenicity islands.
In vitro secretion of a heterologous protein into the surrounding medium using a targeting polypeptide.
Enhanced protein secretion using a defined medium composition.
Documented Applications
In vitro expression and secretion of a heterologous protein of interest using a recombinant Salmonella strain with a T3SS and a targeting polypeptide.
Production of secreted proteins using secretion fractions and downstream purification of proteins produced by T3SS-directed secretion [procedural detail omitted for safety].
Use of virulence/pathogenicity-reducing gene and pathogenicity island deletions to create safer recombinant Salmonella strains for protein production [procedural detail omitted for safety].
Use of kits or culture formats that include a recombinant Salmonella strain and defined medium for protein production [procedural detail omitted for safety].
Secretion of diverse protein cargos including enzymes, biomaterials, antibodies/scFvs, and rhGH [procedural detail omitted for safety].
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