Pangenome reconstruction of Lactobacillaceae metabolism predicts species-specific metabolic traits.

Strains across the Lactobacillaceae family form the basis for a trillion-dollar industry. Our understanding of the genomic basis for their key traits is fragmented, however, including the metabolism that is foundational to their industrial uses. Pangenome analysis of publicly available Lactobacillaceae genomes allowed us to generate genome-scale metabolic network reconstructions for 26 species of industrial importance. Their manual curation led to more than 75,000 gene-protein-reaction associations that were deployed to generate 2,446 genome-scale metabolic models. Cross-referencing genomes and known metabolic traits allowed for manual metabolic network curation and validation of the metabolic models. As a result, we provide the first pangenomic basis for metabolism in the Lactobacillaceae family and a collection of predictive computational metabolic models that enable a variety of practical uses.IMPORTANCELactobacillaceae, a bacterial family foundational to a trillion-dollar industry, is increasingly relevant to biosustainability initiatives. Our study, leveraging approximately 2,400 genome sequences, provides a pangenomic analysis of Lactobacillaceae metabolism, creating over 2,400 curated and validated genome-scale models (GEMs). These GEMs successfully predict (i) unique, species-specific metabolic reactions; (ii) niche-enriched reactions that increase organism fitness; (iii) essential media components, offering insights into the global amino acid essentiality of Lactobacillaceae; and (iv) fermentation capabilities across the family, shedding light on the metabolic basis of Lactobacillaceae-based commercial products. This quantitative understanding of Lactobacillaceae metabolic properties and their genomic basis will have profound implications for the food industry and biosustainability, offering new insights and tools for strain selection and manipulation.

Meta-analysis Driven Strain Design for Mitigating Oxidative Stresses Important in Biomanufacturing.

As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing. This work integrated more than 7000 E. coli adaptive laboratory evolution (ALE) mutations across 59 experiments to statistically associate mutated genes to 2 ROS tolerance ALE conditions from 72 unique conditions. Mutant oxyR, fur, iscR, and ygfZ were significantly associated and hypothesized to contribute fitness in ROS stress. Across these genes, 259 total mutations were inspected in conjunction with transcriptomics from 46 iModulon experiments. Ten mutations were chosen for reintroduction based on mutation clustering and coinciding transcriptional changes as evidence of fitness impact. Strains with mutations reintroduced into oxyR, fur, iscR, and ygfZ exhibited increased tolerance to H2O2 and acid stress and reduced SOS response, all of which are related to ROS. Additionally, new evidence was generated toward understanding the function of ygfZ, an uncharacterized gene. This meta-analysis approach utilized aggregated and interoperable multiomics data sets to identify mutations conferring industrially relevant phenotypes with the least drawbacks, describing an approach for data-driven strain engineering to optimize microbial cell factories.

Using the reconstructed genome-scale human metabolic network to study physiology and pathology.

Metabolism plays a key role in many major human diseases. Generation of high-throughput omics data has ushered in a new era of systems biology. Genome-scale metabolic network reconstructions provide a platform to interpret omics data in a biochemically meaningful manner. The release of the global human metabolic network, Recon 1, in 2007 has enabled new systems biology approaches to study human physiology, pathology and pharmacology. There are currently more than 20 publications that utilize Recon 1, including studies of cancer, diabetes, host-pathogen interactions, heritable metabolic disorders and off-target drug binding effects. In this mini-review, we focus on the reconstruction of the global human metabolic network and four classes of its application. We show that computational simulations for numerous pathologies have yielded clinically relevant results, many corroborated by existing or newly generated experimental data.

A variational principle for computing nonequilibrium fluxes and potentials in genome-scale biochemical networks.

We derive a convex optimization problem on a steady-state nonequilibrium network of biochemical reactions, with the property that energy conservation and the second law of thermodynamics both hold at the problem solution. This suggests a new variational principle for biochemical networks that can be implemented in a computationally tractable manner. We derive the Lagrange dual of the optimization problem and use strong duality to demonstrate that a biochemical analogue of Tellegen's theorem holds at optimality. Each optimal flux is dependent on a free parameter that we relate to an elementary kinetic parameter when mass action kinetics is assumed.

Biomass as a source of chemical feedstocks: an economic evaluation.

It is suggested that the raw materials and technology exist for basing a major fraction of the U.S. chemical industry on four fermentation products, used in the proper portions: ethanol, isopropanol, n-butanol, and 2,3-butanediol. The primary route for introduction of these materials is dehydration of the alcohols and diols to olefins, which would cause little disruption of the existing industry downstream from the olefins. The proposed substitution has the advantages that it would provide a smooth transition toward renewable feedstocks, while decreasing dependence on fossil sources of organic material and use of toxic materials. However, to make these materials attractive as feedstocks or intermediates in chemical production, their current prices must be substantially reduced. Even with the optimum mix, their largeseale utilization will only occur at about 20 to 40 percent of their estimated chemical prices.

Metabolic modeling of microbial strains in silico.

The large volume of genome-scale data that is being produced and made available in databases on the World Wide Web is demanding the development of integrated mathematical models of cellular processes. The analysis of reconstructed metabolic networks as systems leads to the development of an in silico or computer representation of collections of cellular metabolic constituents, their interactions and their integrated function as a whole. The use of quantitative analysis methods to generate testable hypotheses and drive experimentation at a whole-genome level signals the advent of a systemic modeling approach to cellular and molecular biology.

In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data.

A significant goal in the post-genome era is to relate the annotated genome sequence to the physiological functions of a cell. Working from the annotated genome sequence, as well as biochemical and physiological information, it is possible to reconstruct complete metabolic networks. Furthermore, computational methods have been developed to interpret and predict the optimal performance of a metabolic network under a range of growth conditions. We have tested the hypothesis that Escherichia coli uses its metabolism to grow at a maximal rate using the E. coli MG1655 metabolic reconstruction. Based on this hypothesis, we formulated experiments that describe the quantitative relationship between a primary carbon source (acetate or succinate) uptake rate, oxygen uptake rate, and maximal cellular growth rate. We found that the experimental data were consistent with the stated hypothesis, namely that the E. coli metabolic network is optimized to maximize growth under the experimental conditions considered. This study thus demonstrates how the combination of in silico and experimental biology can be used to obtain a quantitative genotype-phenotype relationship for metabolism in bacterial cells.

Growth factor consumption and production in perfusion cultures of human bone marrow correlate with specific cell production.

Perfusion cultures of human bone marrow mononuclear cells (BMMNC) provide a unique in vitro model of hematopoiesis, supporting growth of both accessory and hematopoietic elements. In this study, bioreactors were used to analyze the consumption and production of growth factors (GFs) in relation to each other and to the cells produced. The exogenously added GFs interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and erythropoietin (Epo) each exhibited different, but reproducible, consumption kinetics. Epo and IL-3 were consumed slowly for the first 5-7 days, and then the consumption rate of both increased. Epo consumption reached a plateau by day 10, whereas IL-3 consumption continued to increase. Consumption of SCF was similar to that of Epo, but began 2-3 days earlier. GM-CSF was consumed throughout the culture period in an accelerating manner. Consumption of SCF and Epo were related, because omission of Epo from the growth medium reduced SCF consumption by 53% and omission of SCF reduced Epo consumption by 82%. A reproducible relationship between cumulative GF consumption and total cell production was observed. Epo was most potent, with 5900 molecules consumed per cell produced, whereas 69,400 molecules of SCF were consumed per cell generated. More specifically, Epo consumption was correlated (r = 0.92 and 0.96) with the number of glycophorin A-positive (glyA+) cells produced, and the rate of Epo consumption varied with the progression of cells through the erythroid lineage. Consequently, measurement of GF consumption rates may be useful for quantifying the types of cells present in a culture. Endogenous GF production was also examined. G-CSF and MIP-1 alpha were present at high levels during the first 4 days but then declined rapidly. LIF first appeared in the second week and steadily increased thereafter. Omission of SCF from the medium allowed the detection of endogenous SCF production, and the kinetics was similar to that of LIF. IL-6 production was biphasic, with a peak and decline in week 1 and an increase during week 2. TGF-beta was below the level of detection in these cultures. The results suggest that perfusion supports accessory and hematopoietic elements which interact and therefore represent a partially functional tissue ex vivo. This system provides a useful model for studying relationships within GF networks and for elucidating the conditions that result in primitive cell expansion ex vivo.

Can Dexter cultures support stem cell proliferation?

The in vitro culture of mouse bone marrow (Dexter cultures) has allowed a detailed analysis of the biology of murine hematopoiesis. However, attempts to develop stable long-term human bone marrow cultures have been unsuccessful. Available culture systems all have finite and relatively short lifetimes. The reasons for the limited longevity are unknown. Utilizing computer-assisted integration techniques, we have theoretically simulated culture cell production kinetics to help identify factors that may be responsible for culture decay, as well as to suggest possible means of improving culture longevity. The simulation demonstrates that removal of stem cells is a possible mechanism leading to culture decline. Under the standard bone marrow culture conditions, even with a high stem cell renewal rate, the cultures appear to be destined to fail. Thus, the development of proper sampling techniques or improved stem cell retention may be critical to obtain successful long-term cultures.

Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with burst-forming units-erythroid.

Flow cytometry has been used in recent years to study antigenic and physical changes accompanying hematopoietic cell differentiation. Such studies have provided the basis for rapid and objective assays that are potential alternatives to the colony assays currently in widespread use. In this report, erythropoiesis was examined in growth factor-supplemented perfused cultures of bone marrow mononuclear cells (BMMNC) using flow cytometric analysis of the transferrin receptor (CD71), glycophorin antigen expression occurred with time, Initially, fewer than 10% of the cells were (CD71++, but by day 8, 19-34% of the cells were CD71++gly A-. This was followed by the appearance of gly A on 10-60% of the CD71++ cells. After day 10, CD71 expression on many gly A+ cells decreased so that a population of CD71-gly A+ cells (11-54 accumulated by day 14. Each phenotype was sorted for morphologic identification and colony assay analysis. CD71++gly A- cells were 85% blasts, one-half of which were erythroblasts, and were significantly enriched for burst-forming units-erythroid (BFU-E). The time-varying number of CD71++gly A- cells in these cultures was found to correlate with the number of BF.78-0.97). Three-color analysis was next used to examine CD33 expression on BFU-E, and in fresh BM, most were found to be CD33-. During culture, however, the number of BFU-E recovered from CD33+ populations first increased and then decreased. Therefore, CD33 was not particularly useful for identifying BFU-E. In contrast, CFU-GM were mostly found to be in the CD71+CD33+ population throughout the culture period. When erythropoietin (Epo) was not added to these cultures, the percentage of gly A+ cells was reduced from 33 to 3.3%. Further, the omission of Epo caused an 80% decrease in the number of BFU-E and a corresponding 94% decrease in the number of CD71++gly A- cells, thereby maintaining the relationship between CD71++gly A- cells and BFU-E. Therefore, flow cytometric analysis was found to be useful in assessing erythroid development, and this approach may be used to develop flow cytometric assays for other populations of interest in hematopoietic cell cultures.

Consistent and high rates of gene transfer can be obtained using flow-through transduction over a wide range of retroviral titers.

Flow-through transduction methods have been developed to overcome physical limitations imposed by Brownian motion on retroviral delivery. This method uses net fluid flow of retroviral supernatants through a porous membrane on which the target cells are placed. It is shown that in comparison to static transduction methods, flow-through transductions have the following advantages: (i) flow-through transductions lead to transduction rates that exceed those obtained by static transduction; (ii) flow-through transductions lead to high transduction rates even at low viral concentrations, eliminating many of the concerns associated with the production of high-titer virus supernatants; (iii) flow-through transductions are insensitive to viral titers, eliminating the need to produce consistently retroviral supernatants at given virus concentrations; (iv) flow-through transductions can be carried out without the use of polycations, such as polybrene; and (v) the volume of viral supernatants needed for gene transfer can be sharply reduced. Taken together, these advantages of flow-through transductions are likely to lead to their widespread use for gene transfer work, both in research and clinical settings.

Coupled effects of polybrene and calf serum on the efficiency of retroviral transduction and the stability of retroviral vectors.

The relative concentrations of Polybrene (PB) and calf serum (CS) in retroviral supernatant have considerable effects on the efficiency of retrovirus-mediated gene transfer and the stability of retroviral vectors. The effect of PB on the efficiency of transduction of Moloney murine leukemia virus (MMuLV)-derived vectors is strongly dependent on CS. At a fixed CS concentration, the efficiency of transduction shows a maximum as a function of PB concentration. Increasing the CS concentration shifted this maximum to higher PB concentrations, but the value of the maximum remained the same. Therefore, there were optimal combinations of PB and CS concentrations that maximized the efficiency of gene transfer: 4.4, 8.8, 13.2, and 22 micrograms/ml of PB for 1%, 2.5%, 5%, and 10% (vol/vol) CS, respectively. Moreover, the presence of PB affected significantly the kinetics of retroviral decay. The loss of retroviral activity did not follow simple exponential decay in the absence of PB during the decay period of the viral supernatant. The dynamics of viral inactivation showed an initial phase during which the transduction efficiency remained constant followed by exponential decay. However, in the presence of high PB concentrations (13.2 micrograms/ml) during the decay period of retroviral vectors, the initial delay was lost and the decay was exponential right from the outset. The present results suggest that in addition to virus-cell interactions that occur on the target cell surface, other physico-chemical processes may occur in solution that have profound effect on retroviral activity and therefore they are of particular importance for gene therapy.

Retroviral infection is limited by Brownian motion.

Replication-defective retroviruses are frequently used as gene carriers for gene transfer into target cells. Here we show that the short half-lives of retroviruses limit the distance that they can effectively travel in solution by Brownian motion, and thus the possibility of successful gene transfer. This physiochemical limitation can be overcome, and effective contact between the retroviral gene carrier and the target cell can be obtained, by using net convective flow of retrovirus-containing medium through a layer of target cells. Using model cell lines (NIH-3T3 and CV-1), it was shown that gene transfer rates can be increased by more than an order of magnitude using the same concentration infection medium. High transduction rates could be obtained even in the absence of polycations, such as Polybrene, which heretofore have been required to achieve reasonable transduction rates. This development may play an important role in realizing human gene therapy.

Metabolic flux balance analysis and the in silico analysis of Escherichia coli K-12 gene deletions.

BACKGROUND: Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silico representations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA). FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information. RESULTS: Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coli using FBA and examined the optimal utilization of the E. coli metabolic pathways as a function of environmental variables. We have used an in silico analysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system) essential for aerobic growth of E. coli on glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi-, zwf, and pta- mutant strains were examined in more detail by mapping the capabilities of these in silico isogenic strains. CONCLUSIONS: We found that computational models of E. coli metabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silica results lead to a further understanding of the complex genotype-phenotype relation.

Replication of mini-F plasmids during the bacterial division cycle.

The cell-cycle replication patterns of two mini-F plasmids have been examined using the membrane-elution technique (to produce cells labelled at different times during the division cycle) and scintillation counting (for quantitative analysis of radioactivity incorporated into plasmid DNA). The mini-F plasmid pML31, which contains the oriV and oriS origins of replication, replicates in a cell-cycle-specific manner with a pattern and cell-cycle timing similar to the parental F plasmid. The mini-F plasmid pMF21, deleted for the region containing the oriV origin of replication, replicates more randomly throughout the division cycle. These results suggest that the oriV origin of replication may be related to cell-cycle-specific replication of the F plasmid.

Tissue culture surface characteristics influence the expansion of human bone marrow cells.

Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application.

Examination of serum and bovine serum albumin as shear protective agents in agitated cultures of hybridoma cells.

A murine hybridoma cell line (167.4G5.3) was cultured in batch mode using IMDM containing different serum concentrations and bovine serum albumin (BSA). Cell growth and death, metabolism and antibody production were studied in these cultures. The cells were more susceptible to shear in the stationary and in the decline phase of growth as evidenced by higher death rates. Cell growth was best at high serum concentrations with high specific growth and low specific death rates. When BSA was used instead of serum in IMDM, no protective effect was observed. Cell metabolism and monoclonal antibody production rates were not influenced by the level of serum or by BSA. The use of serum in commercial serum-free media (OPTI-MEM) also resulted in no change in both growth and death rates.

Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production.

A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 10(2) to 10(5) cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 10(6) cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 10(3) cells per ml grew 30% slower than those at 10(4) or 10(5). This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.

Robustness analysis of the Escherichia coli metabolic network.

Genomic, biochemical, and strain-specific data can be assembled to define an in silico representation of the metabolic network for a select group of single cellular organisms. Flux-balance analysis and phenotypic phase planes derived therefrom have been developed and applied to analyze the metabolic capabilities and characteristics of Escherichia coli K-12. These analyses have shown the existence of seven essential reactions in the central metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acid cycle) for the growth in glucose minimal media. The corresponding seven gene products can be grouped into three categories: (1) pentose phosphate pathway genes, (2) three-carbon glycolytic genes, and (3) tricarboxylic acid cycle genes. Here we develop a procedure that calculates the sensitivity of optimal cellular growth to altered flux levels of these essential gene products. The results indicate that the E. coli metabolic network is robust with respect to the flux levels of these enzymes. The metabolic flux in the transketolase and the tricarboxylic acid cycle reactions can be reduced to 15% and 19%, respectively, of the optimal value without significantly influencing the optimal growth flux. The metabolic network also exhibited robustness with respect to the ribose-5-phosphate isomerase, and the ribose-5-phosephate isomerase flux was reduced to 28% of the optimal value without significantly effecting the optimal growth flux. The metabolic network exhibited limited robustness to the three-carbon glycolytic fluxes both increased and decreased. The development presented another dimension to the use of FBA to study the capabilities of metabolic networks.

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.