Isolation and characterization of strong constitutive promoters active in Lactococcus lactis and Escherichia coli

GARAY NOVILLO, J. N.; BARRA, J. L.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión anual SAIB; 2017

Sociedades de Biociencias

Isolation and characterization of strong constitutive promoters functional inLactococcus lactis and Escherichia coli.Lactic acid bacteria are model organisms with an enormous potential for the expression of recombinant proteins for industrial usedue to their GRAS (Generally Recognized As Safe) status combined with the development of biotechnological, genomic and proteomic tools. If the protein to be expressed is non-toxic for the microbial cell factory, constitutive promoters can be used for overexpression. The objective of the present work was to isolate strong constitutive promoters forexpression in bothL. lactisandE. coli. These shuttle promoters allow the construction and preliminary analysis of expression systemsin E. coli, which is a very easy to work bacteria, for later use in lactic acid bacteria. To this end, a library of L. lactis genomic DNA fragments was generated by digestion with different restriction enzymes and subsequently, the 250-3000bp fragments were cloned into pTLGR shuttle vector for promoter analysis where promoter activity was revealed by fluorescent proteins expression (mCherry and GFP). E. coliwere transformed with the ligation mixture and the presence of a DNA fragments with strong constitutive promoter activity were screenedby fluorescent colony phenotype in plateand confirmed by fluorescence microscopy analysis. A plasmid purification was carried out for these colonies and used to transform L. lactis. Colonies showingfluorescent phenotype were selectedand plasmids were purifiedand sequenced. Thanks to the great genetic knowledge of L.lactis, weidentified 4 different DNA regions capable of acting as strong constitutive promoters in both L. lactis andE. coli.Bioinformatic analysis through genomic and transcriptomicsoftwares and databases was carried out to study the putative lactococcalpromoter regions.Preliminary reporter analysis showed that the 4 identified promoter regions areconsiderably more active than one of the most active constitutive promoter described in the literature to date for L. lactisexpression systems.Keywords: strong constitutive promoter, Lactococcus lactis, Escherichia coli