Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium

• Published in: Microorganisms (Basel) Vol. 10; no. 4; p. 777
• Main Authors: Mayer, Janine, Knuuti, Tobias, Baumgarten, Lisa, Menke, Elise, Bischoff, Lena, Bunk, Boyke, Biedendieck, Rebekka
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 05-04-2022; MDPI
• Subjects: Antibiotics; Bacillus; Bacteria; Cloning; Efficiency; genetic tools; Gram-positive bacteria; Libraries; Penicillin; Penicillin G acylase; Peptides; Plasmids; Priestia megaterium; Protein purification; protein secretion; Proteins; recombinant proteins; Screening; Secretion; signal peptide (SP) library; α-Amylase; United States > US; Germany; genetic tools; penicillin G acylase; recombinant proteins; signal peptide (SP) library; Priestia megaterium; protein secretion
• ISSN: 2076-2607; 2076-2607
• DOI: 10.3390/microorganisms10040777

The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins,
provides a promising alternative to common
species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from
was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in
, could be enhanced even up to 29-fold. Overall, only around 170 recombinant
clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for
and also other
species facilitates the identification of suitable SPs for secretion of recombinant proteins.