Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae.

Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two-compartment scale-down chemostat system imposing feast-famine conditions to characterize the long-term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild-type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale-down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well-mixed chemostat. Our results show that S. cerevisiae subjected to long-term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast-famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.

Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p.

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.

The challenge of improved secretory production of active pharmaceutical ingredients in Saccharomyces cerevisiae: a case study on human insulin analogs.

The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. Great attention has been put on improved secretory production of active pharmaceutical ingredients, and the secretory pathway of this eukaryotic host has been the playground of diverse strain engineering studies, aiming at enhanced cellular capacities for folding and trafficking of the target proteins. However, the cellular quality assessment for secretory proteins remains mostly unpredictable, and different target proteins often do not picture similar secretion yields, underlining the dependency of efficient secretion on the physicochemical properties of the protein of interest. In this study, two human insulin analog precursors (IAPs) with minor differences in their amino acid sequences were used as model secretory proteins. No differences between cells expressing these two proteins were found in the IAP transcript levels, gene copy numbers, or intra-cellularly accumulated proteins, yet a more than sevenfold difference in their secretion yields was found. Physiological characterization of cells expressing these proteins in batch processes revealed no significant difference in their specific growth rate, but an altered overflow metabolism. Global transcriptome analysis carried out in chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated and indicated an increased degradation of the IAP with the low secretion yield. In silico protein structure modeling of the IAPs suggested a difference in conformational stability, induced by the amino acid substitution, which most likely resulted in disparity in trafficking through the secretory pathway and thus a large difference in secretion yields.

Long-term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production.

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation.

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers.

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.

Monitoring and control of microbioreactors: an expert opinion on development needs.

This perspective article is based on an expert panel review on microbioreactor applications in biochemical and biomedical engineering that was organized by the M³C (measurement, monitoring, modelling and control) Working Group of the European Section of Biochemical Engineering Science (ESBES) in the European Federation of Biotechnology (EFB). The aim of the panel was to provide an updated view on the present status of the subject and to identify critical needs and issues for furthering the successful development of microbioreactor monitoring and control. This will benefit future bioprocess development and in vitro toxicity testing. The article concludes with a set of recommendations for extended use and further development of microbioreactors.

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses.

BACKGROUND: The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications. RESULTS: The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low P(i) supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter. CONCLUSIONS: Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins.

Backbone cyclic insulin.

Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single-chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C-terminal lysine and proximal N-terminal. The presence of the ring-closing bond and the native insulin disulfide patterns were documented by LC-MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity.