Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2193-2201 ◽  
Author(s):  
Peter Ravn ◽  
José Arnau ◽  
Søren M. Madsen ◽  
Astrid Vrang ◽  
Hans Israelsen
Keyword(s):  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Protein Production ◽  
Heterologous Protein ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Core ◽  
Transposon Insertion ◽  
Charged Amino Acids ◽  
Secretion Efficiency ◽  
Positively Charged

The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

scholarly journals Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

2001 ◽  
Vol 67 (9) ◽  
pp. 4119-4127 ◽  
Author(s):  
Y. Le Loir ◽  
S. Nouaille ◽  
J. Commissaire ◽  
L. Brétigny ◽  
A. Gruss ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Heterologous Protein ◽  
Staphylococcal Nuclease ◽  
Heterologous Proteins ◽  
Heterologous Protein Secretion ◽  
Precursor Proteins ◽  
Secretion Efficiency

ABSTRACT Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SPNuc) by that of L. lactis protein Usp45 (SPUsp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SPUsp than when directed by SPNuc. This SPUsp effect on Nuc secretion is not due to a better antifolding activity, since SPUsp:Nuc precursor proteins display enzymatic activity in vitro, while SPNuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SPUsp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

scholarly journals The C Terminus of Foamy Retrovirus Gag Contains Determinants for Encapsidation of Pol Protein into Virions

2008 ◽  
Vol 82 (21) ◽  
pp. 10803-10810 ◽  
Author(s):  
Eun-Gyung Lee ◽  
Maxine L. Linial
Keyword(s):  
Site Directed Mutagenesis ◽  
Rna Packaging ◽  
Interaction Domain ◽  
Virus Particles ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Foamy Viruses ◽  
Substitution Mutations ◽  
Gag Assembly ◽  
Positively Charged

ABSTRACT Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.

Lactococcus lactis as a live vector: Heterologous protein production and DNA delivery systems

2011 ◽  
Vol 79 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Daniela Santos Pontes ◽  
Marcela Santiago Pacheco de Azevedo ◽  
Jean-Marc Chatel ◽  
Philippe Langella ◽  
Vasco Azevedo ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Production ◽  
Heterologous Protein ◽  
Dna Delivery ◽  
Delivery Systems ◽  
Heterologous Protein Production

scholarly journals Membrane trafficking of the bacterial adhesin GspB and the accessory Sec transport machinery

2018 ◽  
Vol 294 (5) ◽  
pp. 1502-1515 ◽  
Author(s):  
Cierra Spencer ◽  
Barbara A. Bensing ◽  
Nagendra N. Mishra ◽  
Paul M. Sullam
Keyword(s):  
Signal Peptide ◽  
Membrane Trafficking ◽  
Lipid Membranes ◽  
Large Cell ◽  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Hydrophobic Core ◽  
Gram Positive Bacteria ◽  
Anionic Lipids

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are large, cell wall–anchored adhesins that mediate binding to many host cells and proteins and are associated with bacterial virulence. SRR glycoproteins are exported to the cell surface by the accessory Sec (aSec) system comprising SecA2, SecY2, and 3–5 additional proteins (Asp1 to Asp5) that are required for substrate export. These adhesins typically have a 90-amino acid-long signal peptide containing an elongated N-region and a hydrophobic core. Previous studies of GspB (the SRR adhesin ofStreptococcus gordonii) have shown that a glycine-rich motif in its hydrophobic core is essential for selective, aSec-mediated transport. However, the role of this extended N-region in transport is poorly understood. Here, using protein–lipid co-flotation assays and site-directed mutagenesis, we report that the N-region of the GspB signal peptide interacts with anionic lipids through electrostatic forces and that this interaction is necessary for GspB preprotein trafficking to lipid membranes. Moreover, we observed that protein–lipid binding is required for engagement of GspB with SecA2 and for aSec-mediated transport. We further found that SecA2 and Asp1 to Asp3 also localize selectively to liposomes that contain anionic lipids. These findings suggest that the GspB signal peptide electrostatically binds anionic lipids at the cell membrane, where it encounters SecA2. After SecA2 engagement with the signal peptide, Asp1 to Asp3 promote SecA2 engagement with the mature domain, which activates GspB translocation.

scholarly journals Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum

2021 ◽  
Vol 12 ◽  
Author(s):  
Patrick J. Bakkes ◽  
Patrick Lenz ◽  
Carolin Müller ◽  
Astrid Bida ◽  
Doris Dohmen-Olma ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Fluorescent Protein ◽  
Specific Activity ◽  
Secretory Protein ◽  
Site Directed Mutagenesis ◽  
Optimization Approach ◽  
Hydrophobic Core ◽  
Reporter Strain

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

scholarly journals Controlled Production of Stable Heterologous Proteins in Lactococcus lactis

2002 ◽  
Vol 68 (6) ◽  
pp. 3141-3146 ◽  
Author(s):  
A. Miyoshi ◽  
I. Poquet ◽  
V. Azevedo ◽  
J. Commissaire ◽  
L. Bermudez-Humaran ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Production ◽  
Heterologous Protein ◽  
Nonstructural Protein ◽  
Inducible Promoter ◽  
Staphylococcal Nuclease ◽  
Poor Quality ◽  
Heterologous Proteins ◽  
Wild Type ◽  
Bovine Rotavirus

ABSTRACT The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

scholarly journals Characterization of a Lactococcus lactis promoter for heterologous protein production

2018 ◽  
Vol 17 ◽  
pp. 86-92 ◽  
Author(s):  
Christian E. Ogaugwu ◽  
Qiuying Cheng ◽  
Annabeth Fieck ◽  
Ivy Hurwitz ◽  
Ravi Durvasula
Keyword(s):  
Lactococcus Lactis ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production

scholarly journals Inactivation of theybdDGene in Lactococcus lactis Increases the Amounts of Exported Proteins

2012 ◽  
Vol 78 (19) ◽  
pp. 7148-7151 ◽  
Author(s):  
E. Morello ◽  
S. Nouaille ◽  
N. G. Cortes-Perez ◽  
S. Blugeon ◽  
L. F. C. Medina ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Protein Production ◽  
Insertional Mutagenesis ◽  
Heterologous Protein ◽  
Heterologous Protein Production ◽  
Reporter Strain ◽  
Content Type ◽  
Regulatory Processes ◽  
Production Host

ABSTRACTRandom insertional mutagenesis performed on aLactococcus lactisreporter strain led us to identifyL. lactis ybdDas a protein-overproducing mutant. In different expression contexts, theybdDmutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknown regulatory processes that play a role during protein secretion.

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