Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis.
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.
Ravn
Peter
P
Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark.
Arnau
José
J
Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark.
Madsen
Søren M
SM
Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark.
Vrang
Astrid
A
Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark.
Israelsen
Hans
H
Department of Lactic Acid Bacteria, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005285:Fermentation; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease