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.

Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum

“…The result is not surprising since, (i) other groups have used E. coli lac derived promoters like tac and trc to over-express genes in C. glutamicum [15,39,40], and (ii) the sequence of the −10 box of the tac promoter is identical to that of the consensus promoter of C. glutamicum [23]. Interestingly, this promoter may provide another instance for fine tuning gene expression by using different concentrations of the IPTG inducer [41]. Although a GFP based approach to characterize promoters has been previously described by Knoppova and co-workers [16], the vector described does not possess the versatility of the pTGR platform for testing multiple regulatory sequences.…”

Recombinant protein expression in Escherichia coli: advances and challenges

“…This modification avoids the transient non-genetic LacY - phenotype of a fraction of the cells, allowing uniform entry of the inducer lactose. A second modification ( gal + ) permits the full utilization of lactose as an energy source (Menzella et al, 2003). …”

Manufacturing of recombinant therapeutic proteins in microbial systems

“…Furthermore, promoter strength should be tunable and low priced non-toxic inducers should be available for large scale production. Common promoters are lac, tac, trc for induction with lactose or the lactose analog IPTG, [14,15], araBAD under the control of L-arabinose or λpR/pL for induction by temperature shifts [16][17][18][19][20]. For expression of protein complexes or multimeric proteins such as antibodies and fragments thereof, polycistronic or two-cistronic vectors have been developed.…”