Enhanced recombinant protein production in pyruvate kinase mutant of Bacillus subtilis.

Previous work demonstrated that acetate production was substantially lower in pyruvate kinase (pyk) mutant of Bacillus subtilis. The significantly lower acetate production in the pyk mutant is hypothesized to have positive effect on recombinant protein production either by lifting the inhibitory effect of acetate accumulation in the medium or redirecting the metabolic fluxes beneficial to biomass/protein synthesis. In this study, the impact of the pyk mutation on recombinant protein production was investigated. Green fluorescent protein (GFP+) was selected as a model protein and constitutively expressed in both the wild-type strain and a pyk mutant. In batch cultures, the pyk mutant produced 3-fold higher levels of recombinant protein when grown on glucose as carbon source. Experimental measurements and theoretical analysis show that the higher protein yield of the mutant is not due to removal of an acetate-associated inhibition of expression or gene dosage or protein stability but a much lower acetate production in the mutant allows for a greater fraction of carbon intake to be directed to protein synthesis.

Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint.

BACKGROUND: Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production. RESULTS: Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. CONCLUSION: These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the marker's expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.

Pyruvate kinase-deficient Escherichia coli exhibits increased plasmid copy number and cyclic AMP levels.

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.