Michael M. Meagher

High Cell-Density Fermentation

The purpose of this chapter is to educate the reader about the basic equipment and strategies used in fermentations of P. pastoris in both bench-top and pilot-scale operations. A key element in expression of foreign proteins in this yeast is the need for sufficient aeration, which is achieved by proper mixing of the media and by blending gases to control dissolved oxygen content. Automatic pH control is essential for growth and expression in P. pastoris. Finally, fed-batch fermentations require the use of peristaltic pumps and tubing capable of low rates of delivery for the feeding of nutrients and base. Teflon tubing and peristaltic pump adapters are recommended for fed-batch operations. The information in this chapter should enable a reader with little or no experience to perform a high-cell density fermentation of a P. pastoris expression strain. Although most procedures described here are specifically for the BioFlo III (NBS), it should be possible to achieve high expression levels with almost any good-quality fermentor, modified to accommodate this organism.

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.

Opportunities for ionic liquids in recovery of biofuels

Room temperature ionic liquids have potential as extractants in recovery of butyl alcohol from fermentation broth; water solubility in ionic liquid and ionic liquid solubility in water are important factors affecting selectivity of butyl alcohol extraction from aqueous solutions.

Progress in the development of a recombinant vaccine for human hookworm disease: The Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.

Non-Repressing Carbon Sources for Alcohol Oxidase (AOX1) Promoter of Pichia pastoris.

The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1 utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1 utilizing enzymes, especially in a shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress beta-gal production when methanol was used as an inducer in mut- strain of P. pastoris. Our results show that either one of alanine, sorbitol, mannitol or trehalose can be used as a sole carbon and energy source for P. pastoris, although the doubling time on trehalose was very long. Mut- strains growing in media containing trehalose, alanine, sorbitol and mannitol with methanol (0.5%) as an inducing agent expressed as much or higher amount of beta-gal as compared to the mut+ growing in methanol containing media.

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

Fermentation strategies for recombinant protein production inPichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fedbatch fermentations☆

The methylotrophic yeast Pichia pastoris synthesizes high levels of alcohol oxidase from the AOX1 gene during growth on methanol as a carbon source. We have a transcriptional fusion of the lacZ gene to the AOX1 promoter as a model system for investigating recombinant protein production in an alcohol oxidase (aox1, aox2) defective strain. Growth and recombinant protein production with glycerol as the carbon source (fed at various constant feedrates) was studied. A feedrate of 1 g l−1 h−1 was found to be optimum resulting in a specific activity of 8.62 × 104 U mg−1 dry cell. The specific yield did not improve when glycerol was increased in steps. High feeding rates gave low specific yields (U mg−1 dry cell mass) and high cell masses. Low protein yields at higher glycerol feedrates were due to partial repression of the AOX1 promoter by glycerol and the by-product, ethanol. In comparison, the wild type (Mut+) strain gave a maximum specific yield of 5.52 × 104 U mg−1 dry cell.

Improved production of recombinant ovine interferon-τ by Mut+ strain of Pichia pastoris using an optimized methanol feed profile

Recombinant ovine interferon‐τ (r‐oIFN‐τ) production by Pichia pastoris was studied using methanol as the sole carbon source during induction. The cells were grown on glycerol up to a certain cell density before induction of the AOX1 promoter by methanol for expression of the recombinant protein. Cell growth on methanol has been modeled using a substrate‐feed equation, which served as the basis for an effective computer control of the process. The r‐oIFN‐τ concentration in the culture began to decline despite continued cell growth after 50 (± 6) h of induction, which was associated with an increase in proteolytic activity of the fermentation broth. A specific growth rate of 0.025 h‐1 was found to be optimal for r‐oIFN‐τ production. No significant improvement in r‐oIFN‐τ production was observed when the specific growth rate was stepped up before the critical point when r‐oIFN‐τ concentration started decreasing during fermentation. However, best results were obtained when the specific growth rate was stepped down from 0.025 to 0.02 h‐1 at 38 h of induction, whereby the active production period was prolonged until 70 h of induction and the broth protease activity was correspondingly reduced. The corresponding maximum protein yield was 391.7 mg·L‐1 after 70 h of fermentation. The proteolytic activity could be reduced by performing fermentations at specific growth rates of 0.025 h‐1 or below. The recombinant protein production can be performed at an optimal yield by directly controlling the methanol feed rate by a computer‐controlled model. The production profile of r‐oIFN‐τ was found to be significantly different from other secreted and intracellular recombinant protein processes, which is an indication that recombinant protein production in Pichia pastoris needs to be optimized as individual processes following established principles.

Production and Purification of the Heavy Chain Fragment C of Botulinum Neurotoxin, Serotype A, Expressed in the Methylotrophic Yeast Pichia pastoris

A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(H(C)) production while minimizing proteolytic degradation. Concentration of rBoNTA(H(C)) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The H(C) fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The H(C) fragment was purified to >95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified H(C) fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin.

Optimization of temperature-glycerol-pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris.

Abstract This study was undertaken to determine the optimum pH, temperature and glycerol feed rate for the production of recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (rAcAP-5) by Pichia pastoris using response surface methodology (RSM). A central composite design was used as an experimental design for allocation of treatment combinations in three blocks. The variables selected for study were pH, temperature and glycerol feed rate. pH was the most important variable affecting yield, specific yield and specific activity of rAcAP-5. Glycerol feed rate had a significant effect on the specific activity of rAcAP-5 (% of total secreted protein) while temperature did not have a significant effect on the responses. The data showed a trend that gave maximum responses and there was no blocking effect on the responses. The RSM formulated three second order polynomial empirical models relating to the responses. From these models it was possible to determine the optimum conditions variables for maximum yield of rAcAP-5 (1.2 g l−1), the maximum specific yield of rAcAP-5 (11.5 mg g−1 dry cell) and the maximum specific activity of rAcAP-5 (96% of total secreted protein).