A theoretical evaluation of growth yields of yeasts

Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower in S. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors: --S. cerevisiae had a lower protein content than C. utilis; --uptake of glucose by C. utilis requires energy whereas in S. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C. utilis is 35% higher than in S. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth of S. cerevisiae on glucose was 16 g biomass.mol ATP-1. In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways: --via the assumption that the gap is independent of the growth substrate (i.e. a fixed amount of ATP bridges the difference between the theoretical and experimental values). --alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.     

Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production. The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium. Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1. This is much higher than previously reported for batch cultures. Addition of acetic acid or propionic acid lowered the YATP. A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added. This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.     

The NADP(H) redox couple in yeast metabolism

Theoretical calculations of the NADPH requirement for biomass formation indicate that in yeasts this parameter is strongly dependent on the carbon and nitrogen sources used for growth. Enzyme surveys of NADPH-generating metabolic pathways and radiorespirometric studies demonstrate that in yeasts the HMP pathway is the major source of NADPH. Furthermore, radiorespirometric data suggest that in yeasts the HMP pathway activities are close to the theoretical minimum. It may be concluded that the mitochondrial NADPH oxidation, which in yeasts may yield ATP, is quantitatively not an important process.The inability of C. utilis to utilize the NADH produced in formate oxidation as an extra source of NADPH strongly suggests that transhydrogenase activity is absent. Furthermore, the absence of xylose utilization under anaerobic conditions in most facultatively fermentative yeasts indicates that also in these organisms transhydrogenase activity is absent. This conclusion is supported by the observation that anaerobic xylose utilization is observed only in those yeasts which possess a high activity of an NADH-linked xylose reductase. Hence in these organisms the redox-neutral conversion of xylose to ethanol is possible, since the second step in xylose metabolism is mediated by an NAD⁺-linked xylitol dehydrogenase.This paper is adapted from a treatise by the same author, entitled: The NADP(H) redox couple in yeast metabolism, that was awarded the Kluyver prize 1986 by the Netherlands Society of Microbiology     

Application of bioenergetics to modelling the microbial conversion of D-xylose to 2,3-butanediol

During the oxygen limiting growth of Klebsiella oxytoca, the xylose metabolism may be considered as consisting of three components: conversion to 2,3-butanediol by "fermentation," oxidation to carbon dioxide by respiration, and assimilation to cell mass. The amount of energy required for the assimilation of cell mass is assumed to determine the extent to which the two energy producing reactions occur. The activity of each energy producing pathway is also determined by the availability of oxygen and by the energy yield of each pathway. These relationships can be quantified by equating the ATP required for growth and maintenance to the ATP produced by the energy producing reactions. The resulting equation for butanediol production appears similar to the Luedeking and Piret model where the parameters alpha and beta are related to the maximum cell yield from ATP and the maintenance energy requirement. These parameters were estimated from 14 batch fermentations, and the resulting simulation was used to describe the effects of the oxygen transfer rate and the initial xylose concentration on the yields and rates of the 2,3-butanediol fermentation.     

Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures.

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation of glucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass.mol ATP-1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP.mol of glucose-1. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP.mol of glucose-1, or more correctly an energy gain of 0.28 mol of ATP.mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+.mol of malate-1(depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactate -/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate -/H+ symport, though a low-affinity malate- uniport was also implicated. The measured electrical component (delta psi) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.     

Flux balance analysis of photoautotrophic metabolism

Photosynthesis is the principal process responsible for fixation of inorganic carbon dioxide into organic molecules with sunlight as the energy source. Potentially, many chemicals could be inexpensively produced by photosynthetic organisms. Mathematical modeling of photoautotrophic metabolism is therefore important to evaluate maximum theoretical product yields and to deeply understand the interactions between biochemical energy, carbon fixation, and assimilation pathways. Flux balance analysis based on linear programming is applied to photoautotrophic metabolism. The stoichiometric network of a model photosynthetic prokaryote, Synechocystis sp. PCC 6803, has been reconstructed from genomic data and biochemical literature and coupled with a model of the photophosphorylation processes. Flux map topologies for the hetero-, auto-, and mixotrophic modes of metabolism under conditions of optimal growth were determined and compared. The roles of important metabolic reactions such as the glyoxylate shunt and the transhydrogenase reaction were analyzed. We also theoretically evaluated the effect of gene deletions or additions on biomass yield and metabolic flux distributions.     

Growth energetics of an alkaline serine protease-producing strain of Bacillus clausii during continuous cultivation

Glucose-limited chemostats were used to determine the growth yields of biomass of Bacillus clausii PP 473-8 producing an alkaline serine protease Savinase (Novozymes A/S, Bagsværd, Denmark) and a low yield of biomass on oxygen was observed. The energy metabolism was investigated further by setting up simple stoichiometric models for growth on glucose and citrate. In order to determine the parameters in the models, a macromolecular biomass composition was determined based on measured values of protein and RNA combined with literature data. From the macromolecular composition of the biomass the theoretical co-factor and building block requirements needed for biomass formation were calculated. Using the stoichiometric models and data for growth on glucose and citrate the amount of ATP needed for biomass synthesis was estimated to 42.0 mmol ATP/gDW, the P/O ratio to 0.68 and the ATP maintenance to 2.93 mmol ATP/gDW/h. From these values it is concluded that the high oxygen consumption compared with other Bacillus species is due to a low efficiency in respiration resulting in a low P/O ratio. Finally, the energetic parameters were estimated for different architectures of the respiratory chain.     

Thermodynamic evaluation of energy metabolism in mixed substrate catabolism: modeling studies of stationary and oscillatory states

Thermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S(1)), in the presence of a second substrate, formate (S(2)), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S(2) allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S(1) which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S(1)) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (eta) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S(2)/S(1) ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of eta was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high eta predicting that higher biomass yields may be obtained through a variable, high, eta by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Energy-dependence of the Assimilatory Nitrate Uptake in Azorhizobium caulinodans Strain IRBG 46

Nitrate assimilation by suspensions of Azorhizobium caulinodans strain IRBG 46, as determined by disappearance of nitrate ions from the external medium, displayed the requirement of readily utilizable carbon source. Nitrate uptake was blocked by the uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl hydrazone and by an inhibitor of ATPase, N, N — dicyclohexyl carbodiimide. The inhibition of nitrate assimilation in the absence of appropriate carbon source was not overcome by the non-physiological terminal electron donor ascorbate plus N-methyl phenazinium methyl sulphate, a substrate combination that allows electron transfer to O₂ without the synthesis of ATP. These data suggest that transport of nitrate into the cell is directly dependent on ATP.     

In search of a thermodynamic description of biomass yields for the chemotrophic growth of microorganisms

Correlations for the prediction of biomass yields are valuable, and many proposals based on a number of parameters (Y(ATP), Y(Ave), eta(o), Y(c), Gibbs energy efficiencies, and enthalpy efficiencies) have been published. This article critically examines the properties of the proposed parameters with respect to the general applicability to chemotrophic growth systems, a clear relation to the Second Law of Thermodynamics, the absence of intrinsic problems, and a requirement of only black box information. It appears that none of the proposed parameters satisfies all these requirements. Particularly, the various energetic efficiency parameters suffer from major intrinsic problems. However, this article will show that the Gibbs energy dissipation per amount of produced biomass (kJ/C-mod) is a parameter which satisfies the requirements without having intrinsic problems. A simple correlation is found which provides the Gibbs energy dissipation/C-mol biomass as a function of the nature of the C-source (expressed as the carbon chain length and the degree of reduction). This dissipation appears to be nearly independent of the nature of the electron acceptor (e.g., O(2), No(3) (-), fermentation). Hence, a single correlation can describe a very wide range of microbial growth systems. In this respect, Gibbs energy dissipation is much more useful than heat production/C-mol biomass, which is strongly dependent on the electron acceptor used. Evidence is presented that even a net heat-uptake can occur in certain growth systems.The correlation of Gibbs energy dissipation thus obtained shows that dissipation/C-mol biomass increases for C-sources with smaller chain length (C(6) --> C(1)), and increases for both higher and lower degrees of reduction than 4. It appears that the dissipation/C-mol biomass can be regarded as a simple thermodynamic measure of the amount of biochemical "work" required to convert the carbon source into biomass by the proper irreversible carbon-carbon coupling and oxidation/reduction reactions. This is supported by the good correlation between the theoretical ATP requirement for biomass formation on different C-sources and the dissipation values (kJ/C-mol biomass) found. The established correlation for the Gibbs energy dissipation allows the prediction of the chemotrophic biomass yield on substrate with an error of 13% in the yield range 0.01 to 0.80 C-mol biomass/(C)-mol substrate for aerobic/anaerobic/denitrifying growth systems.     

YATP value in Candida tropicalis grown on n-alkanes, fatty acids, and acetate.

The amount of ATP produced during n-alkane, fatty acid, or acetate metabolism in Candida tropicalis has been established from the P/O ratios measured on isolated mitochondria, yield on substrate and carbon balance. For these three kinds of substrates YATP value has been found to be close to 4, although Ysub on acetate is very different from those found with n-alkanes or fatty acids.     

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.     

Biochemical limits to microbial growth yields: An analysis of mixed substrate utilization

A theoretical analysis has been made of carbon conversion efficiency during heterotrophic microbial growth. The expectation was that the maximal growth yield occurs when all the substrate is assimilated and the net flow of carbon through dissimilation is zero. This, however, is not identical to a 100% carbon conversion, since assimilatory pathways lead to a net production of CO(2). It can be shown that the amount of CO(2) produced by way of assimilatory processes is dependent upon the nature of the carbon source, but independent of its degree of reduction and varies between 12 and 29% of the substrate carbon. An analysis of published yield data reveals that nearly complete assimilation can occur during growth on substrates with a high energy content. This holds for substrates with a heat of combustion of ca. 550 kJ/mol C, or a degree of reduction higher than 5 (e.g. ethane, ethanol, and methanol). Complete assimilation can also be achieved on substrates with a lower energy content, provided that an auxiliary energy source is present that cannot be used as a carbon source. This is evident from the cell yields reported for Candida utilis grown on glucose plus formate and for Thiobacillus versutus grown on acetate plus thiosulfate. This evaluation of the carbon conversion efficiency during assimilation also made it possible to compare the energy content of the auxiliary energy substrate added with the quantity of the carbon source it had replaced. It will be shown that utilization of the auxiliary energy source may lead to extreme changes in the efficiency of dissimilatory processes.     

Energetics of bacterial growth: balance of anabolic and catabolic reactions.

Biomass formation represents one of the most basic aspects of bacterial metabolism. While there is an abundance of information concerning individual reactions that result in cell duplication, there has been surprisingly little information on the bioenergetics of growth. For many years, it was assumed that biomass production (anabolism) was proportional to the amount of ATP which could be derived from energy-yielding pathways (catabolism), but later work showed that the ATP yield (YATP) was not necessarily a constant. Continuous-culture experiments indicated that bacteria utilized ATP for metabolic reactions that were not directly related to growth (maintenance functions). Mathematical derivations showed that maintenance energy appeared to be a growth rate-independent function of the cell mass and time. Later work, however, showed that maintenance energy alone could not account for all the variations in yield. Because only some of the discrepancy could be explained by the secretion of metabolites (overflow metabolism) or the diversion of catabolism to metabolic pathways which produced less ATP, it appeared that energy-excess cultures had mechanisms of spilling energy. Bacteria have the potential to spill excess ATP in futile enzyme cycles, but there has been little proof that such cycles are significant. Recent work indicated that bacteria can also use futile cycles of potassium, ammonia, and protons through the cell membrane to dissipate ATP either directly or indirectly. The utility of energy spilling in bacteria has been a curiosity. The deprivation of energy from potential competitors is at best a teleological explanation that cannot be easily supported by standard theories of natural selection. The priming of intracellular intermediates for future growth or protection of cells from potentially toxic end products (e.g., methylglyoxal) seems a more plausible explanation.     

Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.     

Comparative physiology of two protozoan parasites, Leishmania donovani and Trypanosoma brucei, grown in chemostats.

Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate. L. donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T. brucei is 1.06 day-1 and the Ks is 0.06 mM. At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity. L. donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP). Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium. L. donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted. T. brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates. Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism. The Ks is constant as long as glucose is the rate-limiting substrate. The results of this study demonstrate that L. donovani and T. brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.     

The mixed culture of microalgae Chlorella pyrenoidosa and yeast Yarrowia lipolytica for microbial biomass production

Microbial biomass which mostly generated from the microbial processes of bacteria, yeasts, and microalgae is an important resource. Recent concerns in microbial biomass production field, especially microbial lipid production for biofuel, have been focused towards the mixed culture of microalgae and yeast. To more comprehensive understanding of the mixed culture for microbial biomass, mono Chlorella pyrenoidosa, mono Yarrowia lipolytica and the mixed culture were investigated in the present work. Results showed that the mixed culture achieved significantly faster cell propagation of microalga and yeast, smaller individual cell size of yeast and higher relative chlorophyll content of microalga. The mixed culture facilitated the assimilation of carbon and nitrogen and drove the carbon flow to carbohydrate. Besides higher lipid yield (0.77 g/L), higher yields of carbohydrates (1.82 g/L), protein (1.99 g/L) and heating value (114.64 kJ/L) indicated the microbial biomass harvested from the mixed culture have more potential utilization in renewable energy, feedstuff, and chemical industry.

     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

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

Commercial production of heterologous proteins by yeasts has gained considerable interest. Expression systems have been developed forSaccharomyces 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^–1) 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.