title: Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer.
abstract: 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.
authors.affiliation: Microbiology, IBR, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina.
authors.forename: Hugo G
authors.initials: HG
authors.lastname: Menzella
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Ceccarelli
authors.forename: Hugo C
authors.initials: HC
authors.lastname: Gramajo
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D007763:Lac Operon / Q000235:genetics; D007785:Lactose / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007785:Lactose