Metabolic Flux Ratio Analysis Research Articles

Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from 13C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-13C]- and [4-13C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer 13C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.

Metabolic and physiological perturbations of Escherichia coli W3100 by bacterial small RNA RyhB

RyhB is a key regulator of iron level in Escherichia coli (E. coli), which assists in conserving iron for life-sustaining cellular functions when cytoplasmic levels of the ferrous form of iron is limited. RyhB affects glucose metabolism. Seventy percent of the genes that are regulated by RyhB are related to metabolism. We demonstrated for the first time that the activity of the pentose phosphate pathway increased upon ryhB activation using a13C stable isotope-based technique called METAFoR (Metabolic flux ratio analysis). U-13C glucose-based studies showed that the reversible exchange activity of serine and glycine was enhanced by flux redistribution, which further favors NADPH formation. In addition, Entner–Doudoroff (ED) pathway activity was inhibited in the ryhB-defective cells. Quantitative physiology-based experiments highlighted a significant increase in the levels of reactive oxygen species (ROS) in ryhB-induced W3100 E. coli cells in batch culture. A simultaneous decrease in NADH/NAD+ and NADPH/NADP+ ratios outlined the potentially direct roles of NADH and NADPH in antagonizing the excess ROS formed after ryhB activation. Our observations offer a new perspective regarding the roles of RyhB and highlight that this small RNA can significantly affect cell metabolism in addition to its role as a regulator of gene expression.

Central carbon metabolism influences cellulase production in Bacillus licheniformis.

Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering.

13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis ofLactococcus lactisreveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of how L. lactis regulates central metabolism under various conditions.

SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis.

Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses.

Metabolic flux ratio analysis and cell staining suggest the existence of C4 photosynthesis in Phaeodactylum tricornutum.

Mechanisms for carbon fixation via photosynthesis in the diatom Phaeodactylum tricornutum Bohlin were studied recently but there remains a long-standing debate concerning the occurrence of C4 photosynthesis in this species. A thorough investigation of carbon metabolism and the evidence for C4 photosynthesis based on organelle partitioning was needed. In this study, we identified the flux ratios between C3 and C4 compounds in P. tricornutum using (13)C-labelling metabolic flux ratio analysis, and stained cells with various cell-permeant fluorescent probes to investigate the likely organelle partitioning required for single-cell C4 photosynthesis. Metabolic flux ratio analysis indicated the C3/C4 exchange ratios were high. Cell staining indicated organelle partitioning required for single-cell C4 photosynthesis might exist in P. tricornutum. The results of (13)C-labelling metabolic flux ratio analysis and cell staining suggest single-cell C4 photosynthesis exists in P. tricornutum. This study provides insights into photosynthesis patterns of P. tricornutum and the evidence for C4 photosynthesis based on (13)C-labelling metabolic flux ratio analysis and organelle partitioning.

Photorespiration participates in the assimilation of acetate in Chlorella sorokiniana under high light.

The development of microalgae on an industrial scale largely depends on the economic feasibility of mass production. High light induces productive suspensions during cultivation in a tubular photobioreactor. Herein, we report that high light, which inhibited the growth of Chlorella sorokiniana under autotrophic conditions, enhanced the growth of this alga in the presence of acetate. We compared pigments, proteomics and the metabolic flux ratio in C. sorokiniana cultivated under high light (HL) and under low light (LL) in the presence of acetate. Our results showed that high light induced the synthesis of xanthophyll and suppressed the synthesis of chlorophylls. Acetate in the medium was exhausted much more rapidly in HL than in LL. The data obtained from LC-MS/MS indicated that high light enhanced photorespiration, the Calvin cycle and the glyoxylate cycle of mixotrophic C. sorokiniana. The results of metabolic flux ratio analysis showed that the majority of the assimilated carbon derived from supplemented acetate, and photorespiratory glyoxylate could enter the glyoxylate cycle. Based on these data, we conclude that photorespiration provides glyoxylate to speed up the glyoxylate cycle, and releases acetate-derived CO2 for the Calvin cycle. Thus, photorespiration connects the glyoxylate cycle and the Calvin cycle, and participates in the assimilation of supplemented acetate in C. sorokiniana under high light.

Biohydrogen production via the interaction of nitrogenase and anaerobic mixed-acid fermentation in marine bacteria

Biohydrogen production that utilizes high-salt organic wastewater as substrate not only could treat organic waste but also could generate green energy—hydrogen. Investigation of the hydrogen-producing mechanism will lay the foundation for the industrialization of biohydrogen production. In this study, analysis revealed that mixed-acid fermentation occurred in Pantoea agglomerans. However, the amount of hydrogen that would be produced by mixed-acid fermentation (50%) was found to be less than the actual total hydrogen content in the fermenter (67.4%). Hence, a proteomic study was conducted to detect other possible pathways of hydrogen production. The results showed a nitrogenase participated in hydrogen production as a hydrogenase under anaerobic conditions. The results of both proteomics and 13C metabolic flux ratio analysis proved the substrates consumed during hydrogen production were supplied by EMP and PPP. All of these results revealed that hydrogen production was generated by the interaction of mixed-acid fermentation and nitrogenase in the marine bacterium P. agglomerans. EMP and PPP provided pyruvate, NAD(P)H, and ATP for hydrogen production.

Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress

The ability of a microorganism to adapt to changes in the environment, such as in nutrient or oxygen availability, is essential for its competitive fitness and survival. The cellular objective and the strategy of the metabolic response to an extreme environment are therefore of tremendous interest and, thus, have been increasingly explored. However, the cellular objective of the complex regulatory structure of the metabolic changes has not yet been fully elucidated and more details regarding the quantitative behaviour of the metabolic flux redistribution are required to understand the systems-wide biological significance of this response. In this study, the intracellular metabolic flux ratios involved in the central carbon metabolism were determined by fractional (13)C-labeling and metabolic flux ratio analysis (MetaFoR) of the wild-type E. coli strain JM101 at an oxidative environment in a chemostat. We observed a significant increase in the flux through phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (MEZ) and serine hydroxymethyltransferase (SHMT). We applied an ε-constraint based multi-objective optimization to investigate the trade-off relationships between the biomass yield and the generation of reductive power using the in silico iJR904 genome-scale model of E. coli K-12. The theoretical metabolic redistribution supports that the trans-hydrogenase pathway should not play a direct role in the defence mounted by E. coli against oxidative stress. The agreement between the measured ratio and the theoretical redistribution established the significance of NADPH synthesis as the goal of the metabolic reprogramming that occurs in response to oxidative stress. Our work presents a framework that combines metabolic flux ratio analysis and multi-objective optimization to investigate the metabolic trade-offs that occur under varied environmental conditions. Our results led to the proposal that the metabolic response of E. coli to paraquat-induced oxidative stress is globally conserved and coordinated.

Integration of in vivo and in silico metabolic fluxes for improvement of recombinant protein production

The filamentous fungus Aspergillus niger is an efficient host for the recombinant production of the glycosylated enzyme fructofuranosidase, a biocatalyst of commercial interest for the synthesis of pre-biotic sugars. In batch culture on a minimal glucose medium, the recombinant strain A. niger SKAn1015, expressing the fructofuranosidase encoding suc1 gene secreted 45 U/mL of the target enzyme, whereas the parent wild type SKANip8 did not exhibit production. The production of the recombinant enzyme induced a significant change of in vivo fluxes in central carbon metabolism, as assessed by 13C metabolic flux ratio analysis. Most notably, the flux redistribution enabled an elevated supply of NADPH via activation of the cytosolic pentose phosphate pathway (PPP) and mitochondrial malic enzyme, whereas the flux through energy generating TCA cycle was reduced. In addition, the overall possible flux space of fructofuranosidase producing A. niger was investigated in silico by elementary flux mode analysis. This provided theoretical flux distributions for multiple scenarios with differing production capacities. Subsequently, the measured flux changes linked to improved production performance were projected into the in silico flux space. This provided a quantitative evaluation of the achieved optimization and a priority ranked target list for further strain engineering. Interestingly, the metabolism was shifted largely towards the optimum flux pattern by sole expression of the recombinant enzyme, which seems an inherent attractive property of A. niger. Selected fluxes, however, changed contrary to the predicted optimum and thus revealed novel targets—including reactions linked to NADPH metabolism and gluconate formation.

TCA cycle activity in Saccharomyces cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates.

Metabolic responses of Saccharomyces cerevisiae to different physical and chemical environmental conditions were investigated in glucose batch culture by GC-MS-detected mass isotopomer distributions in proteinogenic amino acids from (13)C-labelling experiments. For this purpose, GC-MS-based metabolic flux ratio analysis was extended from bacteria to the compartmentalized metabolism of S. cerevisiae. Generally, S. cerevisiae was shown to have low catabolic fluxes through the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. Notably, respiratory TCA cycle fluxes exhibited a strong correlation with the maximum specific growth rate that was attained under different environmental conditions, including a wide range of pH, osmolarity, decoupler and salt concentrations, but not temperature. At pH values of 4.0 to 6.0 with near-maximum growth rates, the TCA cycle operated as a bifurcated pathway to fulfil exclusively biosynthetic functions. Increasing or decreasing the pH beyond this physiologically optimal range, however, reduced growth and glucose uptake rates but increased the 'cyclic' respiratory mode of TCA cycle operation for catabolism. Thus, the results indicate that glucose repression of the TCA cycle is regulated by the rates of growth or glucose uptake, or signals derived from these. While sensing of extracellular glucose concentrations has a general influence on the in vivo TCA cycle activity, the growth-rate-dependent increase in respiratory TCA cycle activity was independent of glucose sensing.

High-throughput metabolic flux analysis based on gas chromatography–mass spectrometry derived 13C constraints

13C-constrained flux balancing analysis based on gas chromatography–mass spectrometry data is presented here as a simple and robust method for the estimation of intracellular carbon fluxes. In this approach, the underdetermined system of metabolite balances deduced from stoichiometric relations and measured extracellular rates is complemented with 13C constraints from metabolic flux ratio analysis. Fluxes in central carbon metabolism of exponentially growing Escherichia coli were estimated by 13C-constrained flux balancing from three different 13C-labeled glucose experiments. The best resolution of the network was achieved using 13C constraints derived from [U- 13C]glucose and [1- 13C]glucose experiments. The corresponding flux estimate was in excellent agreement with a solution that was independently obtained with a comprehensive isotopomer model. This new methodology was also demonstrated to faithfully capture the intracellular flux distribution in E. coli shake flasks and 1-ml deep-well microtiter plates. Due to its simplicity, speed, and robustness, 13C-constrained metabolic flux balancing is promising for routine and high-throughput analysis on a miniaturized scale.

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS.

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13C] and [U-13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.

Metabolic flux responses to pyruvate kinase knockout in Escherichia coli.

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA.

Blocking glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (Pgi) in Escherichia coli led to a greatly reduced maximum specific growth rate. Examination of the operational catabolic pathways and their flux ratios using [U-(13)C(6)]glucose-labeling experiments and metabolic flux ratio analysis provide evidence for the pentose phosphate (PP) pathway as the primary route of glucose catabolism in the knock-out mutant. The resulting extensive flux through the PP pathway disturbs apparently the reducing power balance, since overexpression of the recently identified soluble transhydrogenase UdhA improves significantly the growth rate of the Pgi mutant. The presented results provide first evidence that UdhA restores the cellular redox balance by catalyzing electron transfer from NADPH to NADH.

Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA

Blocking glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (Pgi) in Escherichia coli led to a greatly reduced maximum specific growth rate. Examination of the operational catabolic pathways and their flux ratios using [U-13C6]glucose-labeling experiments and metabolic flux ratio analysis provide evidence for the pentose phosphate (PP) pathway as the primary route of glucose catabolism in the knock-out mutant. The resulting extensive flux through the PP pathway disturbs apparently the reducing power balance, since overexpression of the recently identified soluble transhydrogenase UdhA improves significantly the growth rate of the Pgi mutant. The presented results provide first evidence that UdhA restores the cellular redox balance by catalyzing electron transfer from NADPH to NADH.

Large-scale prediction of phenotype: concept.

The capability to gather organism wide data has far outstripped the ability to understand it. Transforming large-scale data into a "better" cell requires tools that integrate physiology with its environment. One such tool is large-scale mathematical models that marry stoichiometry and kinetics with metabolic regulation and control. It is straightforward to determine stoichiometry (at least for central pathways), and kinetics can be roughly approximated where need be. However, the molecular details of the "metabolic wiring" managing the cell are often missing. Presented here is a surrogate for these missing details based on a simple premise; over evolutionary time, biological systems have developed objective-based programs that frugally manage gene expression and enzyme activity. Mathematically, this notion can be represented as sets of nonlinear control or "management" problems which, when solved in parallel with the model balances, offer a prediction of how gene expression and enzyme activity are modulated, in the absence of specific mechanistic details. We present a model of Escherichia coli central carbon metabolism, describing batch aerobic growth on glucose, in which transcription, translation, and activity of the gene products of 45 genes is "managed" using this approach. The model consists of 122 species (metabolites, enzymes, mRNA pools, and biomass) and describes 46 reactions (17 reversible). The model is identified (kinetic parameters as well as management structure) from metabolic flux ratio (METAFoR) analysis and physiological measurements. Simulations of a pyruvate kinase knockout strain are compared with experiments and it is shown the model is capable of accurately capturing the metabolic reprogramming resulting from the deletion. Analysis of the mRNA expression pattern, translational pattern and enzyme activity pattern of the wild-type versus mutant indicates a combination of expression and specific activity shifts are responsible for observed differences. While being only a first step toward large-scale physiological modeling, this work is important in two ways. First, it strengthens the hypothesis that unknown mechanism can be reasonably approximated using objective-based management criteria. Second, it provides a dynamic means to couple large-scale analysis technologies with physiology at the single-gene, single-protein level.

Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism.

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

Bioreaction Network Topology and Metabolic Flux Ratio Analysis by Biosynthetic Fractional 13C Labeling and Two-Dimensional NMR Spectroscopy

Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.