Physiological and technological aspects of large-scale heterologous-protein production with yeasts

Abstract

Commercial production of heterologous proteins by yeasts has gained considerable interest. Expression systems have been developed for Saccharomyces cerevisiae and a number of other yeasts. Generally, much attention is paid to the molecular aspects of heterologous-gene expression. The success of this approach is indicated by the high expression levels that have been obtained in shake-flask cultures. For large-scale production however, possibilities and restrictions related to host-strain physiology and fermentation technology also have to be considered. In this review, these physiological and technological aspects have been evaluated with the aid of numerical simulations. Factors that affect the choice of a carbon substrate for large-scale production involve price, purity and solubility. Since oxygen demand and heat production (which are closely linked) limit the attainable growth rate in large-scale processes, the biomass yield on oxygen is also a key parameter. Large-scale processes impose restrictions on the expression system. Many promoter systems that work well in small-scale systems cannot be implemented in industrial environments. Furthermore, large-scale fed-batch fermentations involve a substantial number of generations. Therefore, even low expression-cassette instability has a profound effect on the overall productivity of the system. Multicopy-integration systems may provide highly stable expression systems for industrial processes. Large-scale fed-batch processes are typically performed at a low growth rate. Therefore, effects of a low growth rate on the physiology and product formation rates of yeasts are of key importance. Due to the low growth rates in the industrial process, a substantial part of the substrate carbon is expended to meet maintenance-energy requirements. Factors that reduce maintenance-energy requirements will therefore have a positive effect on product yield. The relationship between specific growth rate and specific product formation rate (kg product-[kg biomass]-1.h-I) is the main factor influencing production levels in large-scale production processes. Expression systems characterized by a high specific rate of product formation at low specific growth rates are highly favourable for large-scale heterologous-protein production.

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

“…For this reason, development of efficient processes for the production of recombinant proteins using microorganisms has become important. A key step in this production is selection of a promoter that directs expression of the target gene in the host strain selected (11). Hence, promoter strength and tight regulation (a low basal level of expression yet a high conditional level of expression) that can be achieved easily and cost-effectively are key goals (14).…”

“…Phosphate-responsive promoters have been exploited in other microorganisms, such as E. coli (6,18) and S. cerevisiae (27). However, a growth limitation other than sugar limitation in E. coli and S. cerevisiae triggers acetic or alcoholic fermentation, leading to arrest of cell growth and lower productivity (11,28). In addition to this problem, Hensing et al (11) indicated that difficulties in maintaining phosphate limitation in complex media and switching to a phosphate-free medium in large-scale fermentation severely limited the applicability of phosphate-responsive promoters.…”

“…However, a growth limitation other than sugar limitation in E. coli and S. cerevisiae triggers acetic or alcoholic fermentation, leading to arrest of cell growth and lower productivity (11,28). In addition to this problem, Hensing et al (11) indicated that difficulties in maintaining phosphate limitation in complex media and switching to a phosphate-free medium in large-scale fermentation severely limited the applicability of phosphate-responsive promoters. The results of this study showing that P. pastoris does not produce ethanol at a level that may cause arrest of cell growth or lower productivity even with a high glucose concentration offer hope that the roadblocks may be overcome.…”

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

“…This organism is traditionally used for large-scale production of baker's yeast and ethanol, with a considerable increase in the production of fuel ethanol in the last 3 decades (4,6,50). Additionally, this platform has been successfully applied to the production of valuable heterologous proteins on an industrial scale (26,35), such as various FDA-approved pharmaceuticals, including insulin (34) and hepatitis B surface antigen (23). Yeast-based expression systems excel because of their available constitutive or strongly inducible promoters and their growth to high cell densities on inexpensive substrates.…”

Auxotrophic Yeast Strains in Fundamental and Applied Research

“…After genetic modification, marker genes may be applied to counterselect against revertants. Especially during gene expression from episomal vectors, this counterselection is often a prerequisite for avoiding overgrowth of the population with cells that have lost the expression vector (18,33).…”