Growth on Formic Acid Is Dependent on Intracellular pH Homeostasis for the Thermoacidophilic Methanotroph Methylacidiphilum sp. RTK17.1

Members of the genus Methylacidiphilum, a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5–3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h−1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation.

Impact of Altered Trehalose Metabolism on Physiological Response of Penicillium chrysogenum Chemostat Cultures during Industrially Relevant Rapid Feast/Famine Conditions

Due to insufficient mass transfer and mixing issues, cells in the industrial-scale bioreactor are often forced to experience glucose feast/famine cycles, mostly resulting in reduced commercial metrics (titer, yield and productivity). Trehalose cycling has been confirmed as a double-edged sword in the Penicillium chrysogenum strain, which facilitates the maintenance of a metabolically balanced state, but it consumes extra amounts of the ATP responsible for the repeated breakdown and formation of trehalose molecules in response to extracellular glucose perturbations. This loss of ATP would be in competition with the high ATP-demanding penicillin biosynthesis. In this work, the role of trehalose metabolism was further explored under industrially relevant conditions by cultivating a high-yielding Penicillium chrysogenum strain, and the derived trehalose-null strains in the glucose-limited chemostat system where the glucose feast/famine condition was imposed. This dynamic feast/famine regime with a block-wise feed/no feed regime (36 s on, 324 s off) allows one to generate repetitive cycles of moderate changes in glucose availability. The results obtained using quantitative metabolomics and stoichiometric analysis revealed that the intact trehalose metabolism is vitally important for maintaining penicillin production capacity in the Penicillium chrysogenum strain under both steady state and dynamic conditions. Additionally, cells lacking such a key metabolic regulator would become more sensitive to industrially relevant conditions, and are more able to sustain metabolic rearrangements, which manifests in the shrinkage of the central metabolite pool size and the formation of ATP-consuming futile cycles.

Microbial Community Decision Making Models in Batch and Chemostat Cultures

Microbial community simulations using genome scale metabolic networks (GSMs) are relevant for many application areas, such as the analysis of the human microbiome. Such simulations rely on assumptions about the culturing environment, affecting if the culture may reach a metabolically stationary state with constant microbial concentrations. They also require assumptions on decision making by the microbes: metabolic strategies can be in the interest of individual community members or of the whole community. However, the impact of such common assumptions on community simulation results has not been investigated systematically. Here, we investigate four combinations of assumptions, elucidate how they are applied in literature, provide novel mathematical formulations for their simulation, and show how the resulting predictions differ qualitatively. Crucially, our results stress that different assumption combinations give qualitatively different predictions on microbial coexistence by differential substrate utilization. This fundamental mechanism is critically under explored in the steady state GSM literature with its strong focus on coexistence states due to crossfeeding (division of labor).

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum. In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi → 2 pyruvate + 5 NTP + Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi → ADP + PPi. Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids. IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.

Contribution of Complex I NADH Dehydrogenase to Respiratory Energy Coupling in Glucose-Grown Cultures of Ogataea parapolymorpha

ABSTRACT The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h−1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations. IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.

Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining.

Respiratory Physiology of Lactococcus lactis in Chemostat Cultures and Its Effect on Cellular Robustness in Frozen and Freeze-Dried Starter Cultures

ABSTRACT In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D = 0.1 h−1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rates to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate, and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10% of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D = 0.1 h−1 were less robust after freeze-drying and had lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells. IMPORTANCE Lactococcus lactis is used in large quantities by the food and biotechnology industries. L. lactis can use oxygen for respiration if heme is supplied in the growth medium. This has been extensively studied in batch cultures using various mutants, but quantitative studies of how the cell growth affects respiratory metabolism, energetics, and cell quality are surprisingly scarce. Our results demonstrate that the respiratory metabolism of L. lactis is remarkably flexible and can be modulated by controlling the specific growth rate. We also link the physiological state of cells during cultivation to the quality of frozen or freeze-dried cells, which is relevant to the industry that may lack understanding of such relationships. This study extends our knowledge of respiratory metabolism in L. lactis and its impact on frozen and freeze-dried starter culture products, and it illustrates the influence of cultivation conditions and microbial physiology on the quality of starter cultures.