INVESTIGADORES
MENZELLA Hugo Gabriel
artículos
Título:
Novel Escherichia coli strain allows efficient recombinant protein production using lactose as inducer
Autor/es:
MENZELLA H.G.; CECCARELLI E.; GRAMAJO H.
Revista:
BIOENGINEERING AND BIOTECHNOLOGY
Editorial:
JOHN WILEY & SONS INC
Referencias:
Año: 2003 vol. 82 p. 809 - 817
ISSN:
0006-3592
Resumen:
An important characteristic of promoters used in recombinant protein
production in Escherichi coli is their inducibility in a simple and
cost-effective manner. The IPTG inducible promoters lac, tac, and trc
are powerful and widely used for basic research. However, the use of
IPTG in large-scale production is undesirable due to its high cost and
toxicity. The promoters mentioned above can also be induced by the
addition of lactose, which has the double role of inducer and carbon and
energy source. Nevertheless, the use of this sugar in industrial
processes has several drawbacks, which result in low volumetric yields
and difficulties in process control. We have genetically engineered a
BL21 strain to allow the efficient use of lactose as inducer in
fed-batch cultures. Two modifications were introduced, the exchange of
the wild-type lac operator by a constitutive one (lacO(c)) and the
replacement of the gal alleles to recover the Gal(+) phenotype. The
constitutive expression of the lac operon overcame the negative effects
that the Lac nongenetic heterogeneity of wild-type E. coli introduces
when lactose is used as inducer. The gal(+) genotype allowed the
complete use of the lactose as carbon and energy source. The relevance
of these two modifications in the efficient utilization of lactose as
inducer was demonstrated in fed-batch cultures of the novel recombinant
strain MP101 harboring expression vectors containing the calf
prochymosin gene or the pccB gene, which encodes for the
carboxyltransferase component of a propionyl-CoA carboxylase complex
from Streptomyces coelicolor. Similar levels of recombinant protein
production (up to 16 g/L) were obtained by using either lactose or IPTG
as inducers, which confirmed the success of the genetics modifications
introduced.