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

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.

Effects of NaCl on gene expression of agmatine deiminase pathway genes of putrescine inLactobacillus delbrueckiiandEscherichia coli

“…The results of the present study showed that expression of aguR is generally low and it was not affected by NaCl. Similarly, other studies showed that aguR expression is not affected by agmatine concentration (Linares, Alvarez-Sieiro, et al, 2015;, glucose concentration (Linares et al, 2013), or the pH of the culture medium (Del Rio, Linares, et al, 2016;. As shown in Figures 4 and 5, the culture pH of the two strains followed a similar trend at different NaCl concentrations.…”

“…The results of the present study showed that expression of aguR is generally low and it was not affected by NaCl. Similarly, other studies showed that aguR expression is not affected by agmatine concentration (Linares, Alvarez‐Sieiro, et al, 2015; Linares, del Rio, et al, 2015), glucose concentration (Linares et al, 2013), or the pH of the culture medium (Del Rio, Linares, et al, 2016; Del Rio, Redruello, et al, 2016). The low expression of aguR presumably ensures it is correctly located at the cell surface where it can act as an agmatine sensor.…”

“…The low expression of aguR presumably ensures it is correctly located at the cell surface where it can act as an agmatine sensor. Agmatine can lead to the polymerization of the AguR protein, and the aguB promoter then drives transcription of the catabolic genes to produce putrescine by combining with carboxyl compounds (Linares, Alvarez‐Sieiro, et al, 2015). NaCl might affect agmatine pathway induction and the subsequent polymerization of AguR, consequently affecting AguB activation.…”

Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model

“…The most commonly used expression systems in L. lactis , as well as in other LAB, includes P170 ( Madsen et al, 1999 ), P (Zn)zitR ( Llull and Poquet, 2004 ), XIES (Xylose-Inducible Expression System) ( Miyoshi et al, 2004 ), NICE (Nisin Controlled Expression System) ( Mierau and Kleerebezem, 2005 ), SICE (Stress-Inducible Controlled Expression System) ( Benbouziane et al, 2013 ), Zirex (Zinc-Regulated Expression System) ( Mu et al, 2013 ), and ACE (Agmatine Controlled Expression System) ( Linares et al, 2015a ).…”

Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

“…Various systems have been developed, of which the NIsin-Controlled Expression system (NICE) is mostly used [12]. Also other systems such as Zirex (Zinc-regulated expression system) [13] and ACE (agmatine-controlled expression) [14] were developed that can be used alone or in combination to enable sequential expression. To introduce foreign DNA into L. lactis, electroporation is currently the golden standard.…”