Download Tipo de Comunicación: Comunicación Oral Simposio

Tipo de Comunicación:
Comunicación Oral
Simposio:
BIOINGENIERÍA MOLECULAR, BIOLOGÍA DE SISTEMAS E INGENIERÍA METABÓLICA.
NUEVAS TECNOLOGÍAS
Título:
Engineering Lactic Acid Bacteria to improve plasmid DNA and protein production
Autores:
O. D. Duarte Sofia, C. Martins Maria, Andrade Sílvia, M. F. Prazeres Duarte, A. Monteiro
Gabriel
Centro de Trabajo:
iBB - Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto
Superior Técnico, Universidade de Lisboa
Email:
[email protected]
Palabras Clave:
Lactic Acid Bacteria; plasmid copy number; 5’-UTR; pAMβ1 origin of replication; gene
therapy
Comunicación:
Lactic Acid Bacteria (LAB) are very promising hosts for production of plasmid DNA and
recombinant proteins due to their Generally Recognized As Safe status. The traditional host
Escherichia coli produces harmful lipopolysaccharides that can be co-purified with the
product of interest, turning Gram-positive LAB safer alternatives, allowing simpler and more
cost effective downstream procedures. On the contrary, the low copy number of the
available LAB plasmids as well as the presence of a high level of endonuclease activity in
the majority of the strains, are major problems to overcome. Our goal is to develop a highly
efficient LAB/high copy number plasmid platform for the production of pharmaceutical-grade
pDNA to be used in Gene Therapy. In parallel the enhancement of plasmid copy numbers
can be easily adapted for the production of recombinant proteins by LAB. The plasmid copynumber of the shuttle vector pTRKH3 is dependent on the pAMβ1 origin of replication,
which is dependent on its encoded RepE protein. Modifications at the repE 5’-untranslated
region (5’-UTR) can affect both the transcription and the translation rates. Therefore, the
Shine-Dalgarno sequence was modified at different sites, by site-directed mutagenesis, in
order to generate a 5’-UTR that leads to a higher level of production of the RepE protein
and ultimately to an increase in the plasmid copy number in LAB. The analysis of the
biomass and pH profiles during growth showed that modifications in the 5’-UTR sequences
apparently did not interfere with the overall cell metabolism. The data analysis from qPCR
showed an increase of 3.5 fold in the copy number from one of the modified plasmids when
replicating in L. lactis LMG 19460. The levels of repE transcripts are being evaluated by
rtPCR. In order to improve the yield and the quality of the pDNA produced by LAB we are
using a CRISPR/Cas9 system to knockout the nth gene that codes for an endonuclease in
L. lactis LMG 19460 and it is expected that its deletion will minimize non-specific digestion
of DNA.