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

Linares DM, Alvarez-Sieiro P, del Rio B, Ladero V, Redruello B, Martin MC, Fernandez M, Alvarez MA - Microb. Cell Fact. (2015)

Bottom Line: Lactococcus lactis has been safely consumed in fermented foods for millennia.The 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.

View Article: PubMed Central - PubMed

Affiliation: Dairy Research Institute, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Rio Linares s/n, 33300, Villaviciosa, Spain. daniml@ipla.csic.es.

ABSTRACT

Background: Lactococcus 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.

Results: This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). 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.

Conclusion: The 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.

No MeSH data available.


Related in: MedlinePlus

Comparison of PEP activity. a In the NICE system, PEP activity was monitored in L. lactis NZ9000 pNZ8048-pep cells induced at different nisin concentrations for 3 h after reaching OD600, while b in the ACE system, PEP activity was monitored in L. lactis NZ9000 pACE-pep cells induced with different agmatine concentrations (added to the culture medium before inoculation) after 7 h of growth. Data represent the mean of three independent experiments. Bars indicate standard deviations (*p < 0.05)
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Fig4: Comparison of PEP activity. a In the NICE system, PEP activity was monitored in L. lactis NZ9000 pNZ8048-pep cells induced at different nisin concentrations for 3 h after reaching OD600, while b in the ACE system, PEP activity was monitored in L. lactis NZ9000 pACE-pep cells induced with different agmatine concentrations (added to the culture medium before inoculation) after 7 h of growth. Data represent the mean of three independent experiments. Bars indicate standard deviations (*p < 0.05)

Mentions: To confirm the usefulness and efficiency of the ACE system, the pep gene of M. xanthus, which encodes a prolyl-endopeptidase of biomedical interest, was cloned into appropriate plasmids for introduction into L. lactis NZ9000. The resulting L. lactis NZ9000 pACE-pep was induced with different agmatine concentrations (0, 0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 20, 40 and 60 mM) and the PEP activity assayed. No activity was detected in cultures without agmatine, but was observed even with the lowest agmatine concentration tested (0.001 mM). Above this concentration, the induction level increased with the agmatine concentration until 0.1 mM agmatine (21.04 mU mg−1) (no significant increase in PEP activity was obtained with concentrations of >0.1 mM) (Fig. 4). PEP activity was sought in the soluble and insoluble fractions, but was only seen in the former.Fig. 4


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

Linares DM, Alvarez-Sieiro P, del Rio B, Ladero V, Redruello B, Martin MC, Fernandez M, Alvarez MA - Microb. Cell Fact. (2015)

Comparison of PEP activity. a In the NICE system, PEP activity was monitored in L. lactis NZ9000 pNZ8048-pep cells induced at different nisin concentrations for 3 h after reaching OD600, while b in the ACE system, PEP activity was monitored in L. lactis NZ9000 pACE-pep cells induced with different agmatine concentrations (added to the culture medium before inoculation) after 7 h of growth. Data represent the mean of three independent experiments. Bars indicate standard deviations (*p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696319&req=5

Fig4: Comparison of PEP activity. a In the NICE system, PEP activity was monitored in L. lactis NZ9000 pNZ8048-pep cells induced at different nisin concentrations for 3 h after reaching OD600, while b in the ACE system, PEP activity was monitored in L. lactis NZ9000 pACE-pep cells induced with different agmatine concentrations (added to the culture medium before inoculation) after 7 h of growth. Data represent the mean of three independent experiments. Bars indicate standard deviations (*p < 0.05)
Mentions: To confirm the usefulness and efficiency of the ACE system, the pep gene of M. xanthus, which encodes a prolyl-endopeptidase of biomedical interest, was cloned into appropriate plasmids for introduction into L. lactis NZ9000. The resulting L. lactis NZ9000 pACE-pep was induced with different agmatine concentrations (0, 0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 20, 40 and 60 mM) and the PEP activity assayed. No activity was detected in cultures without agmatine, but was observed even with the lowest agmatine concentration tested (0.001 mM). Above this concentration, the induction level increased with the agmatine concentration until 0.1 mM agmatine (21.04 mU mg−1) (no significant increase in PEP activity was obtained with concentrations of >0.1 mM) (Fig. 4). PEP activity was sought in the soluble and insoluble fractions, but was only seen in the former.Fig. 4

Bottom Line: Lactococcus lactis has been safely consumed in fermented foods for millennia.The 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.

View Article: PubMed Central - PubMed

Affiliation: Dairy Research Institute, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Rio Linares s/n, 33300, Villaviciosa, Spain. daniml@ipla.csic.es.

ABSTRACT

Background: Lactococcus 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.

Results: This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). 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.

Conclusion: The 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.

No MeSH data available.


Related in: MedlinePlus