Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis

Aleš Berlec,Borut Štrukelj,Janja Kocjan,Katja Škrlec,Maria Olenic

doi:10.1038/s41598-018-19402-1

Aleš Berlec, Borut Štrukelj + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-19402-1

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Abstract

Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.

Highlights

Lactococcus lactis is a gram-positive lactic acid bacterium that is used widely in the dairy industry[1]
PnisA was amplified from pNZ8148 by PCR and, at the same time, a second multiple cloning site, (MCS2), containing unique restriction recognition sites NdeI, PciI, SacI, XhoI and HindIII was introduced using appropriate primers
A gene fragment (53 bp) containing the ribosome binding site (RBS) and MCS2 and with unique restriction recognition sites was amplified with partially overlapping primers and cloned into pNZ8148m

Summary

Introduction

Lactococcus lactis is a gram-positive lactic acid bacterium that is used widely in the dairy industry[1]. Polycistronic expression usually results in decreased yields of the protein that is further downstream from the promoter This can be improved by the addition of an additional, strong promoter upstream of every recombinant gene[9]. CRISPR-Cas[9] derived genome editing tools have revolutionized the fields of genetics, biology and biotechnology in model eukaryotic organisms[13,14] Despite their bacterial origin, the use of CRISPR-Cas[9] systems is less common in bacteria[13,14,15,16]. The use of CRISPR-Cas[9] systems is less common in bacteria[13,14,15,16] Their applications in bacteria include genome editing, gene regulation, production of generation antimicrobials, DNA imaging, etc.[13,16,17]. CRISPR-Cas[9] was applied in Lactobacillus reuteri for eradication of unmodified transformants following recombineering[20] and for modification of the lactococcal phage genome[21]

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