Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

Daniel M Linares,Patricia Alvarez-Sieiro,Beatriz Del Rio,Maria Fernandez,Victor Ladero,Miguel A Alvarez,Begoña Redruello,Mª Cruz Martin

doi:10.1186/s12934-015-0399-x

Daniel M Linares, Patricia Alvarez-Sieiro + Show 6 more

Open Access

https://doi.org/10.1186/s12934-015-0399-x

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Abstract

BackgroundLactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes.ResultsThis paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter PaguB. The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten.ConclusionThe ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

Highlights

Lactococcus lactis has been safely consumed in fermented foods for millennia
We previously showed that aguR is a regulatory gene encoding a transmembrane protein (AguR) that acts as a one-component signal transduction system able to sense the extracellular agmatine concentration and trigger transcriptional activation of the aguB promoter [34]
To develop an efficient gene expression system, carbon catabolite repression (CCR) had to be inactivated so that glucose could be used as a carbon source

Summary

Introduction

Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bac‐ terium has become of industrial importance as an expression host for the overproduction of lipopolysaccharidefree recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. Safe history of use in dairy fermentations, this bacterium has been classified as a food grade microorganism ‘Generally Recognized As Safe’ (GRAS) by the US Food and Drug Administration (FDA), and has led it to receive ‘Qualified Presumption of Safety’ (QPS) status from the European Food Safety Authority (EFSA) [5]. It is an efficient secretor of extracellular recombinant proteins, has low protease activity

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