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Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida.

Eveline-Marie Lammens¹, Daniel C Volke², Alison Kerremans¹

¹Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462, 3001, Leuven, BE, Belgium.

Communications Biology

|February 5, 2025

Summary

This summary is machine-generated.

A new phi15 bacteriophage expression system offers a stringent and flexible alternative to the T7 platform for Pseudomonas putida. This optimized system enhances protein production yields and is suitable for various biotechnological applications.

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Area of Science:

Microbiology
Synthetic Biology
Biotechnology

Background:

The T7 phage RNA polymerase (RNAP) is a common expression system but causes cytotoxicity in non-model microbes.
Developing alternative expression platforms is crucial for expanding protein production in diverse microbial hosts.

Purpose of the Study:

To develop and optimize a novel phage phi15-based expression system for Pseudomonas putida.
To create a stringent and highly inducible system for enhanced protein production.

Main Methods:

Engineered a phi15 RNAP and orthogonal promoter for controlled gene expression.
Introduced a phi15 lysozyme mutant for tight regulation and a phi15 gp16 inhibitor to decouple growth and production.
Developed optimized P. putida hosts and Golden Gate-compatible vectors.

Main Results:

Achieved a stringent expression system with 200-fold inducibility.
Decoupling cell growth and protein production increased expression levels by 20%.
Demonstrated a 2.5- to 5-fold increase in fluorinase yield compared to existing systems.

Conclusions:

The phi15 expression system provides a versatile and efficient alternative to the T7 system for P. putida.
The system's components (RNAP, promoter, lysozyme, growth-decoupler) offer a flexible 'plug-and-play' solution for microbial biotechnology.