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Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
Published on: March 16, 2012
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Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
Innanurdiani Koko1, Adelene Ai-Lian Song2,3, Mas Jaffri Masarudin1,4
1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
BMC Biotechnology
|November 29, 2019
Summary
This study developed novel integrative vectors for stable heterologous protein production in L. lactis. The engineered systems enable efficient protein secretion and surface display, showing potential for industrial applications and live vaccine delivery.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Site-specific integration enables stable heterologous protein expression in host genomes.
- Lactococcal phage TP901-1 integrating system was utilized to construct integrative vectors.
- Vectors were designed for either secretion or surface display of proteins.
Purpose of the Study:
- To construct and evaluate integrative vectors for heterologous protein secretion and surface display in a lactococcal system.
- To assess the efficacy of different promoter and signal peptide combinations.
- To demonstrate the utility of the system for producing functional proteins.
Main Methods:
- Construction of eight integrative vectors with varying lactococcal promoters (PnisA, P170) and signal peptides (USP45, SPK1).
- Utilized a helper vector carrying the integrase gene for genome integration.
- Employed a nuclease gene as a reporter for secretion and display verification.
- Assessed protein secretion and surface display using Nuc reporter and whole-cell ELISA.
Main Results:
- Successfully constructed and integrated eight vectors into the L. lactis genome.
- Demonstrated secretion and surface display of the Nuc reporter protein.
- Identified SPK1 as a superior signal peptide for Nuc secretion compared to USP45-LEISSTCDA.
- Observed comparable surface display efficiencies, except for P170-USP45 combination.
Conclusions:
- Engineered integrative vectors are suitable for heterologous protein production in lactococcal systems.
- The system holds potential for industrial applications and live vaccine delivery.
- Specific promoter-signal peptide combinations influence protein secretion and display efficiency.
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