Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens.

Jiyeon Park,Benedict-Uy Fabia,Jung-Hoon Ahn,Nguyen-Mihn Hieu,Joshua Bingwa

doi:10.3390/biomedicines9060679

Jiyeon Park, Benedict-Uy Fabia + Show 3 more

Open Access

https://doi.org/10.3390/biomedicines9060679

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Abstract

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

Highlights

Pseudomonas fluorescens is a psychrotrophic, gram-negative bacterium that has shown potential as an expression host. It uses a type I secretion system (T1SS) in the form of its ATP-binding cassette (ABC) transporter, TliDEF [1], which is composed of TliD, TliE, and TliF
We focused on growth factors because of their potential applications in medicine, cosmeceuticals, and stem cell differentiation [19,20,21]
To further verify that P. fluorescens ΔfleQ is a viable protein factory, we introduced six growth factors into the of bacteria, namely, insulin-like growth factor I (IGFI),Factors

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Pseudomonas fluorescens is a psychrotrophic, gram-negative bacterium that has shown potential as an expression host. It uses a type I secretion system (T1SS) in the form of its ATP-binding cassette (ABC) transporter, TliDEF [1], which is composed of TliD, TliE, and TliF. The three components function as the ABC protein, membrane fusion protein, and outer membrane protein, respectively [2]. T1SS, such as TliDEF, bypass the periplasm, allowing the secretion of proteins with diverse sizes [3]. Its simple growth conditions and ability to reach acceptable cell density without optimization make

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