Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno-economically using HIV-1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52-83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 95-102, 2009

The glycerol and methanol concentrations in Pichia pastoris fermentations were measured on-line using Fourier transform infrared spectroscopy and an attenuated total reflection probe. Partial least squares regression was used to obtain calibration models. The models were regressed on synthetic multi-component spectra and semi-synthetic fermentation broth spectra. These were obtained by spectral addition. The accuracy for the on-line measurement of glycerol, given as standard error of prediction (SEP), was determined to 0.68 g/l, and the SEP of methanol was 0.13 g/l. We show how reliable calibration models are obtained relatively effortlessly by replacing extensive sampling from the reactor with simple mathematical manipulations of the model regression spectra.

In the present paper we describe the cloning and extracellular expression of the HIV-1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low-temperature fed-batch strategy for decreasing end-product degradation by proteases. The nef gene in a pPICZalphaA vector was integrated into the genome of three different P. pastoris strains, namely X-33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild-type strain (X-33) was found to be the best choice. The decreased end-product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44-fold utilizing the low-temperature strategy compared with the standard fermentation. Purification of histidine-tagged Nef was performed in one step using a Ni(2+)-nitrilotriacetate-Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix-assisted laser-desorption ionization-time-of-flight MS, reversed-phase HPLC and N-terminal-sequence analysis.

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The purpose of the present study was to ascertain the optimal concentration of dissolved oxygen in order to maximize the intracellular glucose oxidase formation in Aspergillus niger. Cultivations performed in a 3.5 l laboratory reactor showed that a dissolved oxygen concentration at 3% of saturation at a total pressure of 1.2 bar was optimal for maximizing intracellular glucose oxidase activity. Cultivations performed at higher dissolved oxygen concentrations did not produce as much glucose oxidase as those performed at 3%, although the formation rate was high. Experiments revealed that maximal intracellular glucose oxidase formation for the A. niger strain used, is accomplished by limiting the gluconic acid production rate by means of maintaining a low dissolved oxygen concentration. Several attempts to achieve higher intracellular glucose oxidase activity were also made by manipulating the glucose concentration at a 3% dissolved oxygen concentration. However, no enhancement in glucose oxidase activity was observed.