Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A.

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.

Nonenzymatic Glycosylation of Lepidopteran-Active Bacillus thuringiensis Protein Crystals.

We used high-pH anion-exchange chromatography with pulsed amperometric detection to quantify the monosaccharides covalently attached to Bacillus thuringiensis HD-1 (Dipel) crystals. The crystals contained 0.54% sugars, including, in decreasing order of prevalence, glucose, fucose, arabinose/rhamnose, galactose, galactosamine, glucosamine, xylose, and mannose. Three lines of evidence indicated that these sugars arose from nonenzymatic glycosylation: (i) the sugars could not be removed by N- or O-glycanases; (ii) the sugars attached were influenced both by the medium in which the bacteria had been grown and by the time at which the crystals were harvested; and (iii) the chemical identity and stoichiometry of the sugars detected did not fit any known glycoprotein models. Thus, the sugars detected were the product of fermentation conditions rather than bacterial genetics. The implications of these findings are discussed in terms of crystal chemistry, fermentation technology, and the efficacy of B. thuringiensis as a microbial insecticide.