Peptides of interest: Editing of Lactococcus lactis proteolytic system to increase its bioactive potential

ABSTRACT The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), PI and PIII proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) ofLactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37°C, and peptide supply on the transcription of these genes. Only transcription of thepepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription ofprtP1, prtP3, pepC, pepN, and the opp-pepO1operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

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Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

International Dairy Journal ◽

10.1016/s0958-6946(02)00022-5 ◽

2002 ◽

Vol 12 (5) ◽

pp. 447-454 ◽

Cited By ~ 24

Author(s):

P Courtin ◽

M Nardi ◽

U Wegmann ◽

V Joutsjoki ◽

J.C Ogier ◽

...

Keyword(s):

Lactococcus Lactis ◽

Proteolytic System

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Editing of the Proteolytic System of Lactococcus lactis Increases Its Bioactive Potential

Applied and Environmental Microbiology ◽

10.1128/aem.01319-20 ◽

2020 ◽

Vol 86 (18) ◽

Cited By ~ 1

Author(s):

Chenxi Huang ◽

Jan Kok

Keyword(s):

Lactococcus Lactis ◽

Large Scale ◽

Bioactive Peptides ◽

Peptide Pool ◽

Bioactive Peptide ◽

Fermented Foods ◽

Proteolytic System ◽

Healthy Human ◽

Content Type ◽

Dipeptidyl Peptidase 4

ABSTRACT Large-scale mass spectrometry-based peptidomics for bioactive-peptide discovery is relatively unexplored because of challenges in intracellular peptide extraction and small-peptide identification. Here, we present an analytical pipeline for large-scale intracellular peptidomics of Lactococcus lactis. It entails an optimized sample preparation protocol for L. lactis, used as an “enzyme complex” to digest β-casein, an extraction method for its intracellular peptidome, and a peptidomics data analysis and visualization procedure. In addition, we proofread the publicly available bioactive-peptide databases and obtained an optimized database of bioactive peptides derivable from bovine β-casein. We used the pipeline to examine cultures of L. lactis MG1363 and a set of 6 isogenic multiple peptidase mutants incubated with β-casein. We observed a clearly strain-dependent accumulation of peptides with several bioactivities, such as angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl peptidase 4 (DPP-IV)-inhibitory, and immunoregulatory functions. The results suggest that both the number of different bioactive peptides and the bioactivity diversity can be increased by editing the proteolytic system of L. lactis. This comprehensive pipeline offers a model for discovery of bioactive peptides in combination with other proteins and might be applicable to other bacteria. IMPORTANCE Lactic acid bacteria (LAB) are very important for the production of safe and healthy human and animal fermented foods and feed and, increasingly more, in the functional food industry. The intracellular peptidomes of LAB are promising reservoirs of bioactive peptides. We show here that targeted genetic engineering of the peptide degradation pathway allows steering the composition of the peptide pool of the LAB Lactococcus lactis and production of peptides with interesting bioactivities. Our work could be used as a guideline for modifying proteolytic systems in other LAB to further explore their potential as cell peptide factories.

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Proteome Analyses of Heme-Dependent Respiration in Lactococcus lactis: Involvement of the Proteolytic System

Journal of Bacteriology ◽

10.1128/jb.186.6.1648-1657.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1648-1657 ◽

Cited By ~ 58

Author(s):

Karin Vido ◽

Dominique le Bars ◽

Michel-Yves Mistou ◽

Patricia Anglade ◽

Alexandra Gruss ◽

...

Keyword(s):

Lactococcus Lactis ◽

Growth Conditions ◽

Transcriptional Analysis ◽

Rich Medium ◽

Triple Mutant ◽

Proteolytic System ◽

Sugar Fermentation ◽

Absolute Requirement ◽

Static Conditions ◽

Respiratory Conditions

ABSTRACT Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

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