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Prevention of C-terminal autoprocessing of Lactococcus lactis SK11 cell-envelope proteinase by engineering of an essential surface loop.

ABSTRACT: The catalytic domain of the cell-envelope proteinase from Lactococcus lactis SK11 has various inserts, situated in external loops of the catalytic domain, compared with the related subtilisins. Protein engineering was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in close proximity to the substrate-binding region and is susceptible to autoproteolysis. We constructed a deletion mutant which lacks 14 residues of this surface loop and subsequently introduced various insertion cassettes coding either for the original loop with three mutations (E205S/E218T/M219S: triple-mutant proteinase) or for neutral spacers (1, 4, 7 and 16 serine residues). Engineered proteinases were analysed for activity, (auto)processing, and cleavage specificity. The presence of residues 205-219 is shown to be essential for proteolytic activity, as only triple-mutant proteinase retained activity towards casein substrates. The triple-mutant proteinase was found to be defective in C-terminal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered proteolytic specificity or altered accessibility of the processing site. The specificity change appears to be subtle, as only small differences were found between wild-type and triple-mutant proteinase in the breakdown of casein substrates.

SUBMITTER: Bruinenberg PG

PROVIDER: S-EPMC1137323 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis.

Project description:The cell envelope proteinase (CEP) of Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a combination of structure prediction tools to create a structural model for the entire CEP enzyme of Lactococcus lactis. The model has implications for how the bacterium interacts with casein micelles during growth in milk, and it has implications regarding the energetics of the proteolytic system. Our model for the CEP indicates that the catalytic triad is activated through a structural change caused by interaction with the substrate. The CEP of L. lactis might become a useful model for the mode of action for enzymes belonging to the large class of S8 proteinases with a PA (protease associated) domain and a downstream fibronectin like domain.

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Engineering Lactococcus lactis for Increased Vitamin K2 Production.

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Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

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Increased production of folate by metabolic engineering of Lactococcus lactis.

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A rapid and versatile tool for genomic engineering in Lactococcus lactis.

Project description:BackgroundLactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.ResultsA high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h.ConclusionsThe work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.

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Project description:A Lactococcus lactis reporter system suitable to detect cell envelope stress in high-throughput settings was developed by fusing the CesR-regulated promoter of llmg0169 to the gfp(uv) gene. A dot blot assay allowed fast detection of green fluorescent protein (GFP) fluorescence even at low production levels. Unexpectedly, this promoter was also induced by mitomycin C via CesR.

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Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis.

Project description:BACKGROUND:Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system. RESULTS:The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. CONCLUSION:The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

| S-EPMC6882331 | biostudies-literature

Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis.

Project description:The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

| S-EPMC309937 | biostudies-literature

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