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

Innanurdiani Koko,Mas Jaffri Masarudin,Adelene Ai-Lian Song,Raha Abdul Rahim

doi:10.1186/s12896-019-0575-x

Innanurdiani Koko, Mas Jaffri Masarudin + Show 2 more

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https://doi.org/10.1186/s12896-019-0575-x

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Abstract

BackgroundSite-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.ResultsThe 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.ConclusionThe 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.

Highlights

Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein
For the recombination to occur, integrase recognises a short sequence of the site (43 bps) which makes the system applicable in numerous L. lactis strains [29,30,31,32,33,34]. Through utilisation of this established integration system, we developed a variety of integrative vectors for secretion and surface display of targeted proteins and compared their efficiency using nuclease as a reporter
The integrative vectors harbouring nuc were transformed into L. lactis NZ9000 harbouring pNZint, and the positive integrants which would obtain erythromycin resistance were tested for their stability to stay integrated in the Bacteria attachment site (attB) site of the genome

Summary

Introduction

Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. Lactococcus lactis, a lactic acid bacteria (LAB) that has been conventionally known as a starter culture in food fermentations such as cheese and yoghurt, was granted with the Generally Recognized as Safe (GRAS) status by the US FDA. It is the most well studied LAB strain due to its well characterised genome which allows researchers to manipulate this bacterium for desired applications. Over expression of heterologous protein causes metabolic load which burdens the host and affect the protein integrity [8] Due to these limitations, constructions of more stable expression systems were attempted through

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