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

JAVIER N. GARAY NOVILLO; JOSÉ LUIS BARRA

Buenos Aires

Congreso; LIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2017

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Lactic acid bacteria are an increasingly interesting model for the expression of recombinant proteins of industrial interest because of their GRAS (Generally Recognized As Safe) condition. If the protein to be overproduced is non-toxic to the bacterium, constitutive promoters can be used for expression. The objective of the present work was to isolate strong promoters of constitutive expression for L. lactis, also active in E. coli. The advantage of these promoters is that they would allow the construction and preliminary analysis of DNA constructs in 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 400-2000 bp fragments were cloned into a reporter vector for promoter regions. E. coli competent cells were transformed with the ligation mixture and the presence of a DNA fragment with strong constitutive promoter activity was revealed by the appearance of red colonies, as a consequence of the expression of the red fluorescent protein of the reporter vector. A plasmid purification was carried out from these red colonies and used to transform L. lactis competent cells. After 3 days of growth, L. lactis colonies showing a rosaceous color were selected, plasmids purified and sequenced. We identified 4 different DNA regions capable of acting as strong constitutive promoter regions in both, L. lactis and E. coli. The putative promoter regions were identified in the L. lactis genome. Moreover, three of the 4 regions identified were also shown (reported) as strong promoter regions in transcriptomic studies of L. lactis. Preliminary analysis showed that the four identified promoter regions (are) would be considerably more active than the most active constitutive promoter described in the literature to date for L. lactis.