Comprehensive study on Escherichia coli genomic expression: Does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings.

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 DeltaackA-pta, DeltapoxB Deltapppc ppc-p37 for recombinant beta-galactosidase production.

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, beta-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards beta-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.

Comparison of different strategies to reduce acetate formation in Escherichia coli.

E. coli cells produce acetate as an extracellular coproduct of aerobic cultures. Acetate is undesirable because it retards growth and inhibits protein formation. Most process designs or genetic modifications to minimize acetate formation aim at balancing growth rate and oxygen consumption. In this research, three genetic approaches to reduce acetate formation were investigated: (1) direct reduction of the carbon flow to acetate (ackA-pta, poxB knock-out); (2) anticipation on the underlying metabolic and regulatory mechanisms that lead to acetate (constitutive ppc expression mutant); and (3) both (1) and (2). Initially, these mutants were compared to the wild-type E. coli via batch cultures under aerobic conditions. Subsequently, these mutants were further characterized using metabolic flux analysis on continuous cultures. It is concluded that a combination of directly reducing the carbon flow to acetate and anticipating on the underlying metabolic and regulatory mechanism that lead to acetate, is the most promising approach to overcome acetate formation and improve recombinant protein production. These genetic modifications have no significant influence on the metabolism when growing the micro-organisms under steady state at relatively low dilution rates (less than 0.4 h(-1)).