Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a ‘real case’, i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in PAOX1-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new PAOX1 synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.

The current interest in microalgae as a sustainable source of next generation biofuels and other valuable substances is driving exploration of their use as unique biotechnological production systems. To design and optimise appropriate production strategies, the behaviour of particular microalgal species should be well characterised under different culture conditions. Thus, flow cytometric (FCM) methods, which are already well established in environmental and toxicological studies of microalgae, are also useful for analysing the physiological state of microalgae, and have the potential to contribute to the rapid development of feasible bioprocesses. These methods are commonly based on the examination of intrinsic features of individual cells within a population (such as autofluorescence or size). Cells possessing the desired physiological or morphological features, which are detectable with or without fluorescent staining, are counted or isolated (sorted) using an FCM device. The options for implementation of FCM in the development of biotechnological processes detailed in this review are (i) analysing the chemical composition of biomass, (ii) monitoring cellular enzyme activity and cell viability, and (iii) sorting cells to isolate those overproducing the target compound or for the preparation of axenic cultures.

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Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

Health and safety concerns, enhanced quality criteria, and environmental sustainability, have prompted investigations into production using recombinant yeasts as a feasible alternative for isolation of proteins from natural animal or plant sources, as well as for processes utilising either mammalian cell cultures or bacterial systems. An overview of recent research papers and review articles provides readers with a comprehensive insight into the field of next-generation yeast expression systems. Major breakthroughs in recombinant yeast technology linked to Pichia pastoris are (i) the public availability of tools to generate proteins with tailored and highly homogenous N-glycan structures, similar to the forms assembled in humans, (ii) the recent accomplishment of the annotation of its genome sequence, and finally, (iii) the presence of the first few (non-glycosylated) therapeutic proteins in Pichia on the market. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry (e.g., enzymes for chemical synthesis and for the modification/synthesis of pharmaceuticals, drug target proteins used for structural analysis or for high throughput screening, proteins for diagnostics, proteinous biomaterials, vaccines, and therapeutic proteins). Nevertheless, the complexity of protein analysis (monitoring) continues to restrict process development for recombinant products. Drawing on combined expertise in molecular biology and process technology, the Institute of Biotechnology (IBT) at the Zurich University of Applied Science (ZHAW) and its international partners have developed solutions which (i) fully eliminate (or partially reduce) the use of methanol, which is undesirable in high-cell-density and high-productivity processes, (ii) match both strain construction and process design with the target protein characteristics to the benefit of the cells' physiological shape, and (iii) allow multi-gene expressions to be balanced to achieve custom tailored and reproducible protein quality at the level of (engineered) posttranslational modifications. In addition to enabling superior product quality specifications to be achieved with reduced development time, these innovations have helped the industries involved to minimise financial risks and the risk of failure, as well as create an opportunity for (new) drugs with improved functionality at low cost.

During our work, flow cytometry was used for a study of the influence of cultivation conditions on a physiological state, mainly on the viability and vitality of the recombinant Pichia pastoris strain producing ?-galactosidase. The fluorescent dyes propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol were used for the determination of the structural integrity and the membrane potential of Pichia pastoris cells, respectively. First the staining procedures were optimised and adapted for the Pichia pastoris yeast cells and later applied in a lab-scale continuous cultivation. Stained samples were analysed with the flow cytometer PAS III Partec or the epifluorescence microscope Nikon Eclipse E400. The following conditions were found to be optimum for the staining of Pichia pastoris cells: 57 ?g/ml propidium iodide in the sample, 5 min incubation time; 2 ?g/ml bis-(1,3-dibutylbarburic acid) trimethine oxonol in the sample, 20 min incubation time. The optimised staining methods were employed in a study of stress-induced physiological response to change of substrate from glycerol to methanol in a glycerol steady state growing culture of Pichia pastoris yeast. During the first 5 h of the transitional state an accumulation of methanol in the culture broth was accompanied by a decreasing concentration of biomass and an increasing amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol. After the adaptation phase the amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol reached steady levels of 2% and 5%, respectively.

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