Glucose-limited Chemostat Cultures Research Articles

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast-famine cycles in a scale-down system.

While traveling through different zones in large-scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DO gradients, we present a scale-down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state (NADH/NAD+) of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acidand degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h−1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h−1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.

Changes in Oxygen Availability during Glucose-Limited Chemostat Cultivations of Penicillium chrysogenum Lead to Rapid Metabolite, Flux and Productivity Responses.

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.

Saccharomyces cerevisiae tolerance to hyper-osmolarity in aerobic glucose-limited chemostat culture

Different mechanisms contribute to regulate cellular functions in order to cope with threats from physiological stress conditions. As a fundamental response to balance excess water loss and restore turgor, Saccharomyces cerevisiae subjected to increased osmolarity accelerate intracellular glycerol biosynthesis and accumulation as a compatible solute. This study assessed cellular response to sorbitol-induced osmolarity in aerobic glucose-limited chemostat culture at various levels of the glycerol flux. Cell number declined slightly without any substantial increase in dry weight and total protein contents following exposure to 1M sorbitol that lasted for 90 min. On the other hand, total glycerol levels increased over time in different yeast cultivations corresponding with enhanced glycerol-3-phosphate dehydrogenase 1 (GPD1) transcript levels, which led to a steady and intensified protein product. These results support literature evidence of accumulated intracellular glycerol regulation at different levels and further increase curiosity to understand yeast tolerance in various applications including bakery, brewery and wine making or leavening of bread underdifferent osmotic conditions.

Analysis of the proteins secreted by Trichoderma harzianum P49P11 under carbon-limited conditions

The wild type strain Trichoderma harzianum was able to synthesize enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase) in glucose-limited chemostat cultures. Fructose/glucose and sucrose conditions provided low levels of PNPGase activity. To investigate whether under these conditions other enzymes were produced, a shotgun proteomics analysis of their supernatants was performed. The analysis has indicated that the different carbon sources used influenced the amounts of proteins secreted including 1,3-beta-glucanosyltransferase, alpha-1,2-mannosidase, alpha-galactosidase and glucan 1,3-beta-glucosidase. The analysis has also suggested the presence of beta-glucosidase, which could also be represented by PNPGase activity. Intracellular metabolites were quantified during PNPGase production for the condition using 20 g/L of glucose in the feed and differences were observed, indicating that intracellular glucose could be inhibiting PNPGase production. SignificanceThis work shows that sugars such as glucose, fructose/glucose and sucrose can be used as substrates for the continuous synthesis of different enzymes under carbon-limited conditions by Trichoderma harzianum. As far as we know, this is the first work about the continuous synthesis of enzymes under carbon-limited conditions suggesting that different easily assimilated carbon sources can be used to generate different enzymatic cocktails. Each enzyme or uncharacterized protein suggested by shotgun proteomics has the potential to become a promising product for biotechnological applications.

A convenient new method for reproducible fed-batch fermentation of fission yeast Schizosaccharomyces pombe.

Development of an open-loop fed-batch protocol for highly reproducible fermentation of fission yeast that starts from batch cultures instead of glucose-limited aerobic chemostat cultures. A new strategy was employed that consists of an exponential feeding phase followed by a starvation period and then a linear feeding phase. A comparison of several independent fed-batch fermentations of a recombinant fission yeast strain showed that while during the initial phase process parameters such as glucose consumption and CO2 evolution varied considerably as expected, they were much more uniform during the third phase. For instance, the normalized standard deviation of glucose consumption was thirty times higher during the exponential feeding phase of the fermentation that during the linear feeding phase. These data demonstrate the usefulness of the proposed strategy. It is expected that by variation of only two parameters (the total amount of glucose fed in the initial phase and the time frame of the starvation phase) the protocol can easily be adapted to other microbes.

Quantitative Flow Cytometry to Understand Population Heterogeneity in Response to Changes in Substrate Availability in Escherichia coli and Saccharomyces cerevisiae Chemostats.

Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose-Limited Cultivation of Penicillium chrysogenum.

During glucose-limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β-lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady-state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose-limited chemostat cultures at the dilution rates (D) of both 0.03 h-1 and 0.05 h-1 are determined to evaluate the quantitative impact on the physiology of a high-yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h-1 , while this is not the case at D = 0.05 h-1 . In addition, a higher cytosolic redox status (NADH/NAD+ ), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h-1 , which are virtually absent at D = 0.03 h-1 . In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose-limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial-scale scenario.

Quantitative metabolomics and metabolic flux analysis reveal impact of altered trehalose metabolism on metabolic phenotypes of Penicillium chrysogenum in aerobic glucose-limited chemostats

In Penicillium chrysogenum, it has been observed that turnover of storage carbohydrates (trehalose, mannitol, arabitol, erythritol and glycogen) resulting in an extra ATP expenditure might partly account for the reduced penicillin productivity under dynamic cultivation conditions. In this work, Penicillium chrysogenum mutants with altered trehalose metabolism were constructed using the Agrobacterium-mediated transformation method. It was observed that impaired trehalose biosynthesis did not result in growth arrest and change of glucose sensitivity to high glucose levels, but negatively influenced the sporulation. Compared with the original strain, in glucose-limited chemostat cultures, the biomass yield on glucose and energy efficiency were slightly enhanced; however, the penicillin productivity was significantly lowered in the trehalose mutant strains. Comparison with a high-yielding P. chrysogenum strain revealed that the original and mutant strains had a lower glucose uptake capacity but higher intracellular levels of free amino acids. Flux estimates through the central carbon metabolism showed distinctive difference in the upper part of the glycolysis and in the pentose phosphate pathway but comparable flux through the TCA cycle. Combining, the striking phenotypic effects observed in the trehalose mutants of P. chrysogenum indicated that trehalose metabolism plays an important role in metabolic regulation and is central to maintaining higher penicillin productivity under glucose-limited chemostat cultures.

Mapping Salmonella typhimurium pathways using 13C metabolic flux analysis

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.

Physiologic and metabolic characterization of Saccharomyces cerevisiae reveals limitations in the synthesis of the triterpene squalene.

Heterologous synthesis of triterpenoids in Saccharomyces cerevisiae from its native metabolite squalene has been reported to offer an alternative to chemical synthesis and extraction from plant material if productivities can be increased.Here, we physiologically characterized a squalene overproducing S. cerevisiae CEN.PK strain to elucidate the effect of cultivation conditions on the production of this central triterpenoid precursor. The maximum achievable squalene concentration was substantially influenced by nutritional conditions, medium composition and cultivation mode. Batch growth on glucose resulted in minimal squalene accumulation, while squalene only significantly accumulated during ethanol consumption (up to 59 mg/gCDW), probably due to increased acetyl-CoA supply on this carbon source. Likewise, low squalene concentrations were observed in glucose-limited chemostat cultivations and improved up to 8-fold upon increasing the ethanol fraction in the feed. In those experiments, a constant, growth-rate-independent specific squalene accumulation rate (2.2 mg/gCDW/h) was recorded resulting in a maximal squalene loading of 30 mg/gCDW at low dilution rates with longer residence times. Coenzyme A availability was identified as possible bottleneck as increased vitamin concentrations, including the Coenzyme A precursor pantothenate, improved squalene titers in batch and chemostat cultivations. This analysis demonstrates that thorough physiologic characterization of production strains is valuable for the identification of bottlenecks already in early stages of strain development and for guiding further optimization efforts.

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

BackgroundReduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD+ balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.ResultsMulti-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD+-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.ConclusionsThe metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.

A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae.

CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol g biomass-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.

Power input effects on degeneration in prolonged penicillin chemostat cultures: A systems analysis at flux, residual glucose, metabolite, and transcript levels.

In the present work, by performing chemostat experiments at 400 and 600 RPM, two typical power inputs representative of industrial penicillin fermentation (P/V, 1.00 kW/m3 in more remote zones and 3.83 kW/m3 in the vicinity of the impellers, respectively) were scaled-down to bench-scale bioreactors. It was found that at 400 RPM applied in prolonged glucose-limited chemostat cultures, the previously reported degeneration of penicillin production using an industrial Penicillium chrysogenum strain was virtually absent. To investigate this, the cellular response was studied at flux (stoichiometry), residual glucose, intracellular metabolite and transcript levels. At 600 RPM, 20% more cell lysis was observed and the increased degeneration of penicillin production was accompanied by a 22% larger ATP gap and an unexpected 20-fold decrease in the residual glucose concentration (Cs,out ). At the same time, the biomass specific glucose consumption rate (qs ) did not change but the intracellular glucose concentration was about sixfold higher, which indicates a change to a higher affinity glucose transporter at 600 RPM. In addition, power input differences cause differences in the diffusion rates of glucose and the calculated Batchelor diffusion length scale suggests the presence of a glucose diffusion layer at the glucose transporting parts of the hyphae, which was further substantiated by a simple proposed glucose diffusion-uptake model. By analysis of calculated mass action ratios (MARs) and energy consumption, it indicated that at 600 RPM glucose sensing and signal transduction in response to the low Cs,out appear to trigger a gluconeogenic type of metabolic flux rearrangement, a futile cycle through the pentose phosphate pathway (PPP) and a declining redox state of the cytosol. In support of the change in glucose transport and degeneration of penicillin production at 600 RPM, the transcript levels of the putative high-affinity glucose/hexose transporter genes Pc12g02880 and Pc06g01340 increased 3.5- and 3.3-fold, respectively, and those of the pcbC gene encoding isopenicillin N-synthetase (IPNS) were more than twofold lower in the time range of 100-200 hr of the chemostat cultures. Summarizing, changes at power input have unexpected effects on degeneration and glucose transport, and result in significant metabolic rearrangements. These findings are relevant for the industrial production of penicillin, and other fermentations with filamentous microorganisms.

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism.

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29mgL-1). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO2 value, 100% N2), limited (zero pO2 value, 75% of cell needs), and low (pO2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7μm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75gL-1). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica.

Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates.

ABSTRACTThe yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1 to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than the mS estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes.IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grown P. pastoris in a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found that P. pastoris decreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.

Maintenance-energy requirements and robustness of Saccharomyces cerevisiae at aerobic near-zero specific growth rates.

BackgroundSaccharomyces cerevisiae is an established microbial platform for production of native and non-native compounds. When product pathways compete with growth for precursors and energy, uncoupling of growth and product formation could increase product yields and decrease formation of biomass as a by-product. Studying non-growing, metabolically active yeast cultures is a first step towards developing S. cerevisiae as a robust, non-growing cell factory. Microbial physiology at near-zero growth rates can be studied in retentostats, which are continuous-cultivation systems with full biomass retention. Hitherto, retentostat studies on S. cerevisiae have focused on anaerobic conditions, which bear limited relevance for aerobic industrial processes. The present study uses aerobic, glucose-limited retentostats to explore the physiology of non-dividing, respiring S. cerevisiae cultures, with a focus on industrially relevant features.ResultsRetentostat feeding regimes for smooth transition from exponential growth in glucose-limited chemostat cultures to near-zero growth rates were obtained by model-aided experimental design. During 20 days of retentostats cultivation, the specific growth rate gradually decreased from 0.025 h−1 to below 0.001 h−1, while culture viability remained above 80 %. The maintenance requirement for ATP (mATP) was estimated at 0.63 ± 0.04 mmol ATP (g biomass)−1 h−1, which is ca. 35 % lower than previously estimated for anaerobic retentostats. Concomitant with decreasing growth rate in aerobic retentostats, transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes resembled transcriptional regulation patterns observed for anaerobic retentostats. The heat-shock tolerance in aerobic retentostats far exceeded previously reported levels in stationary-phase batch cultures. While in situ metabolic fluxes in retentostats were intentionally low due to extreme caloric restriction, off-line measurements revealed that cultures retained a high metabolic capacity.ConclusionsThis study provides the most accurate estimation yet of the maintenance-energy coefficient in aerobic cultures of S. cerevisiae, which is a key parameter for modelling of industrial aerobic, glucose-limited fed-batch processes. The observed extreme heat-shock tolerance and high metabolic capacity at near-zero growth rates demonstrate the intrinsic potential of S. cerevisiae as a robust, non-dividing microbial cell factory for energy-intensive products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0501-z) contains supplementary material, which is available to authorized users.

Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose-limited chemostat culture.

Currently, research is being focused on the industrial-scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial-scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose-limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild-type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial-scale production of fumaric acid are discussed.

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni2+-dependent enzyme and Saccharomyces cerevisiae does not express native Ni2+-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01µmolmin−1mg protein−1 and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni2+ concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni2+-dependent enzymes can be functionally expressed in S. cerevisiae.