Browsing by Author "Carlson, Ross P."

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Answer Set Programming for Computing Constraints-Based Elementary Flux Modes: Application to Escherichia coli Core Metabolism
(MDPI AG, 2020-12) Mahout, Maxime; Carlson, Ross P.; Peres, Sabine
Elementary Flux Modes (EFMs) provide a rigorous basis to systematically characterize the steady state, cellular phenotypes, as well as metabolic network robustness and fragility. However, the number of EFMs typically grows exponentially with the size of the metabolic network, leading to excessive computational demands, and unfortunately, a large fraction of these EFMs are not biologically feasible due to system constraints. This combinatorial explosion often prevents the complete analysis of genome-scale metabolic models. Traditionally, EFMs are computed by the double description method, an efficient algorithm based on matrix calculation; however, only a few constraints can be integrated into this computation. They must be monotonic with regard to the set inclusion of the supports; otherwise, they must be treated in post-processing and thus do not save computational time. We present aspefm, a hybrid computational tool based on Answer Set Programming (ASP) and Linear Programming (LP) that permits the computation of EFMs while implementing many different types of constraints. We apply our methodology to the Escherichia coli core model, which contains 226×106 EFMs. In considering transcriptional and environmental regulation, thermodynamic constraints, and resource usage considerations, the solution space is reduced to 1118 EFMs that can be computed directly with aspefm. The solution set, for E. coli growth on O2 gradients spanning fully aerobic to anaerobic, can be further reduced to four optimal EFMs using post-processing and Pareto front analysis.
Anti-biofilm properties of chitosan-coated surfaces
(2008-01) Carlson, Ross P.; Taffs, Reed L.; Davison, William Marshall; Stewart, Philip S.
Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria and yeast. Reductions in biofilm viable cell numbers ranging from 95% to 99.9997% were demonstrated for Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans on chitosan-coated surfaces over a 54-h experiment in comparison to controls. For instance, chitosan-coated surfaces reduced S. epidermidis surface-associated growth more than 5.5 10log units (99.9997%) compared to a control surface. As a comparison, coatings containing a combination of the antibiotics minocycline and rifampin reduced S. epidermidis growth by 3.9 10log units (99.99%) and coatings containing the antiseptic chlorhexidine did not significantly reduce S. epidermidis surface associated growth as compared to controls. The chitosan effects were confirmed with microscopy. Using time-lapse fluorescence microscopy and fluorescent-dye-loaded S. epidermidis, the permeabilization of these cells was observed as they alighted on chitosan-coated surfaces. This suggests chitosan disrupts cell membranes as microbes settle on the surface. Chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection.
Artificial consortium demonstrates emergent properties of enhanced cellulosic-sugar degradation and biofuel synthesis
(2020-12) Park, Heejoon; Patel, Ayushi; Hunt, Kristopher A.; Henson, Michael A.; Carlson, Ross P.
Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans (a.k.a. Lachnoclostridium phytofermentans), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli, a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O2, creating an anoxic environment for C. phytofermentans. Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli. E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.
Carbon chain length of biofuel- and flavor-relevant volatile organic compounds produced by lignocellulolytic fungal endophytes changes with culture temperature
(2017-09) Schoen, Heidi R.; Hunt, Kristopher A.; Strobel, Gary A.; Peyton, Brent M.; Carlson, Ross P.
Three fungal endophytes from the genus Nodulisporium were studied for volatile organic compound (VOC) production. All three fungi grew on a wide range of carbon substrates ranging from simple sugars to waste biomass sources. The fungi synthesized a number of long and short-chain VOCs, including eucalyptol; 1-butanol, 3-methyl; 1-octen-3-ol; and benzaldehyde, all with potential applications as biofuel or flavor compounds. As culture temperature decreased, average VOC carbon chain length increased, especially for VOCs associated with fatty acid metabolism. The results provide a template for controlling synthesis of desired VOCs through selection of species and culturing conditions.
Combining Targeted Metabolomic Data with a Model of Glucose Metabolism: Toward Progress in Chondrocyte Mechanotransduction
(2016-01) Salinas, Daniel; Minor, Cody A.; Carlson, Ross P.; McCutchen, Carley N.; Mumey, Brendan M.; June, Ronald K.
Osteoarthritis is a debilitating disease likely involving altered metabolism of the chondrocytes in articular cartilage. Chondrocytes can respond metabolically to mechanical loads via cellular mechanotransduction, and metabolic changes are significant because they produce the precursors to the tissue matrix necessary for cartilage health. However, a comprehensive understanding of how energy metabolism changes with loading remains elusive. To improve our understanding of chondrocyte mechanotransduction, we developed a computational model to calculate the rate of reactions (i.e. flux) across multiple components of central energy metabolism based on experimental data. We calculated average reaction flux profiles of central metabolism for SW1353 human chondrocytes subjected to dynamic compression for 30 minutes. The profiles were obtained solving a bounded variable linear least squares problem, representing the stoichiometry of human central energy metabolism. Compression synchronized chondrocyte energy metabolism. These data are consistent with dynamic compression inducing early time changes in central energy metabolism geared towards more active protein synthesis. Furthermore, this analysis demonstrates the utility of combining targeted metabolomic data with a computational model to enable rapid analysis of cellular energy utilization.
Competitive resource allocation to metabolic pathways contributes to overflow metabolisms and emergent properties in cross-feeding microbial consortia
(2018-04) Carlson, Ross P.; Beck, Ashley E.; Phalak, Poonam; Fields, Matthew W.; Gedeon, Tomas; Hanley, Luke; Harcombe, W. R.; Henson, Michael A.; Heys, Jeffrey J.
Resource scarcity is a common stress in nature and has a major impact on microbial physiology. This review highlights microbial acclimations to resource scarcity, focusing on resource investment strategies for chemoheterotrophs from the molecular level to the pathway level. Competitive resource allocation strategies often lead to a phenotype known as overflow metabolism; the resulting overflow byproducts can stabilize cooperative interactions in microbial communities and can lead to cross-feeding consortia. These consortia can exhibit emergent properties such as enhanced resource usage and biomass productivity. The literature distilled here draws parallels between in silico and laboratory studies and ties them together with ecological theories to better understand microbial stress responses and mutualistic consortia functioning.
Complete enumeration of elementary flux modes through scalable demand-based subnetwork definition
(2014-06) Hunt, Kristopher A.; Folsom, James P.; Taffs, Reed L.; Carlson, Ross P.
Motivation: Elementary flux mode analysis (EFMA) decomposes complex metabolic network models into tractable biochemical pathways, which have been used for rational design and analysis of metabolic and regulatory networks. However, application of EFMA has often been limited to targeted or simplified metabolic network representations due to computational demands of the method. Results: Division of biological networks into subnetworks enables the complete enumeration of elementary flux modes (EFMs) for metabolic models of a broad range of complexities, including genome-scale. Here, subnetworks are defined using serial dichotomous suppression and enforcement of flux through model reactions. Rules for selecting appropriate reactions to generate subnetworks are proposed and tested; three test cases, including both prokaryotic and eukaryotic network models, verify the efficacy of these rules and demonstrate completeness and reproducibility of EFM enumeration. Division of models into subnetworks is demand-based and automated; computationally intractable subnetworks are further divided until the entire solution space is enumerated. To demonstrate the strategyâ€™s scalability, the splitting algorithm was implemented using an EFMA software package (EFMTool) and Windows PowerShell on a 50 node Microsoft high performance computing cluster. Enumeration of the EFMs in a genome-scale metabolic model of a diatom, Phaeodactylum tricornutum, identified ~2 billion EFMs. The output represents an order of magnitude increase in EFMs computed compared with other published algorithms and demonstrates a scalable framework for EFMA of most systems.
Decomposition of complex microbial behaviors into resource-based stress responses
(2008-11) Carlson, Ross P.
Motivation: Highly redundant metabolic networks and experimental data from cultures likely adapting simultaneously to multiple stresses can complicate the analysis of cellular behaviors. It is proposed that the explicit consideration of these factors is critical to understanding the competitive basis of microbial strategies.Results: Wide ranging, seemingly unrelated Escherichia coli physiological fluxes can be simply and accurately described as linear combinations of a few ecologically relevant stress adaptations. These strategies were identified by decomposing the centralmetabolism of E.coli into elementary modes (mathematically defined biochemical pathways) and assessing the resource investment costâ€“ benefit properties for each pathway. The approach capitalizes on the inherent tradeoffs related to investing finite resources like nitrogen into different pathway enzymes when the pathways have varying metabolic efficiencies. The subset of ecologically competitive pathways represented 0.02% of the total permissible pathways. The biological relevance of the assembled strategies was tested against 10 000 randomly constructed pathway subsets. None of the randomly assembled collections were able to describe all of the considered experimental data as accurately as the costbased subset. The results suggest these metabolic strategies are biologically significant. The current descriptions were compared with linear programming (LP)-based flux descriptions using the Euclidean distance metric. The current studyâ€™s pathway subset described the experimental fluxes with better accuracy than the LP results without having to test multiple objective functions or constraints and while providing additional ecological insight into microbial behavior. The assembled pathways seem to represent a generalized set of strategies that can describe a wide range of microbial responses and hint at evolutionary processes where a handful of successful metabolic strategies are utilized simultaneously in different combinations to adapt to diverse conditions.
Differentiation of microbial species and strains in coculture biofilms by multivariate analysis of laser desorption postionization mass spectra
(2013-09) Bhardwaj, C.; Cui, Y.; Hofstetter, T.; Liu, S. Y.; Bernstein, Hans C.; Carlson, Ross P.; Ahmed, M.; Hanley, L.
7.87 to 10.5 eV vacuum ultraviolet (VUV) photon energies were used in laser desorption postionization mass spectrometry (LDPI-MS) to analyze biofilms comprised of binary cultures of interacting microorganisms. The effect of photon energy was examined using both tunable synchrotron and laser sources of VUV radiation. Principal components analysis (PCA) was applied to the MS data to differentiate species in Escherichia coliâ€“Saccharomyces cerevisiae coculture biofilms. PCA of LDPI-MS also differentiated individual E. coli strains in a biofilm comprised of two interacting gene deletion strains, even though these strains differed from the wild type K-12 strain by no more than four gene deletions each out of approximately 2000 genes. PCA treatment of 7.87 eV LDPI-MS data separated the E. coli strains into three distinct groups, two â€œpureâ€ groups, and a mixed region. Furthermore, the â€œpureâ€ regions of the E. coli cocultures showed greater variance by PCA at 7.87 eV photon energies compared to 10.5 eV radiation. This is consistent with the expectation that the 7.87 eV photoionization selects a subset of low ionization energy analytes while 10.5 eV is more inclusive, detecting a wider range of analytes. These two VUV photon energies therefore give different spreads via PCA and their respective use in LDPI-MS constitute an additional experimental parameter to differentiate strains and species.
Direct measurement and characterization of active photosynthesis zones inside wastewater remediating and biofuel producing microalgal biofilms
(2014-03) Bernstein, Hans C.; Kessano, M.; Moll, Karen M.; Smith, Terrence; Gerlach, Robin; Carlson, Ross P.; Miller, Charles D.; Peyton, Brent M.; Cooksey, Keith E.; Gardner, Robert D.; Sims, R. C.
Microalgal biofilm based technologies are of keen interest due to their high biomass concentrations and ability to utilize light and CO2. While photoautotrophic biofilms have long been used for wastewater remediation, biofuel production represents a relatively new and under-represented focus area. However, the direct measurement and characterization of fundamental parameters required for industrial control are challenging due to biofilm heterogeneity. This study evaluated oxygenic photosynthesis and respiration on two distinct microalgal biofilms cultured using a novel rotating algal biofilm reactor operated at field- and laboratory-scales. Clear differences in oxygenic photosynthesis and respiration were observed based on different culturing conditions, microalgal composition, light intensity and nitrogen availability. The cultures were also evaluated as potential biofuel synthesis strategies. Nitrogen depletion was not found to have the same effect on lipid accumulation compared to traditional planktonic microalgal studies. Physiological characterizations of these microalgal biofilms identify fundamental parameters needed to understand and control process optimization.
Effects of recombinant precursor pathway variations on poly[(r)-3-hydroxybutyrate] synthesis in Saccharomyces cerevisiae
(2006-07) Carlson, Ross P.; Srienc, Friedrich
Different recombinant R-3-hydroxybutyryl-CoA (3-HB) synthesis pathways strongly influenced the rate and accumulation of the biopolymer poly[(R)-3-hydroxybutyrate] (PHB) in Saccharomyces cerevisiae. It has been previously shown that expression of the Cupriavidus necator PHB synthase gene leads to PHB accumulation in S. cerevisiae [Leaf, T., Peterson, M., Stoup, S., Somers, D., Srienc, F., 1996. Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiology 142, 110]. This finding indicates that native S. cerevisiae expresses genes capable of synthesizing the correct stereochemical substrate for the synthase enzyme. The effects of variations of 3-HB precursor pathways on PHB accumulation were investigated by expressing combinations of C. necator PHB pathway genes. When only the PHB synthase gene was expressed, the cells accumulated biopolymer to approximately 0.2% of their cell dry weight. When the PHB synthase and reductase gene were co-expressed, the PHB levels increased approximately 18 fold to about 3.5% of the cell dry weight. When the beta-ketothiolase, reductase and synthase genes were all expressed, the strain accumulated PHB to approximately 9% of the cell dry weight which is 45 fold higher than in the strain with only the synthase gene. Fluorescent microscopic analysis revealed significant cell-to-cell heterogeneity in biopolymer accumulation. While the population average for the strain expressing three PHB genes was approximately 9% of the cell dry weight, some cells accumulated PHB in excess of 50% of their cell volume. Other cells accumulated no biopolymer. In addition, the recombinant strain was shown to co-produce ethanol and PHB under anaerobic conditions. These results demonstrate that the technologically important organism S. cerevisiae is capable of accumulating PHB aerobically and anaerobically at levels similar to some bacterial systems. The easily assayed PHB system also creates a convenient means of probing in vivo the presence of intracellular metabolites which could be useful for studying the intermediary metabolism of S. cerevisiae.
Effects of Spatial Localization on Microbial Consortia Growth
(2017-01) Venters, Michael; Carlson, Ross P.; Gedeon, Tomas; Heys, Jeffrey J.
Microbial consortia are commonly observed in natural and synthetic systems, and these consortia frequently result in higher biomass production relative to monocultures. The focus here is on the impact of initial spatial localization and substrate diffusivity on the growth of a model microbial consortium consisting of a producer strain that consumes glucose and produces acetate and a scavenger strain that consumes the acetate. The mathematical model is based on an individual cell model where growth is described by Monod kinetics, and substrate transport is described by a continuum-based, non-equilibrium reaction-diffusion model where convective transport is negligible (e.g., in a biofilm). The first set of results focus on a single producer cell at the center of the domain and surrounded by an initial population of scavenger cells. The impact of the initial population density and substrate diffusivity is examined. A transition is observed from the highest initial density resulting in the greatest cell growth to cell growth being independent of initial density. A high initial density minimizes diffusive transport time and is typically expected to result in the highest growth, but this expected behavior is not predicted in environments with lower diffusivity or larger length scales. When the producer cells are placed on the bottom of the domain with the scavenger cells above in a layered biofilm arrangement, a similar critical transition is observed. For the highest diffusivity values examined, a thin, dense initial scavenger layer is optimal for cell growth. However, for smaller diffusivity values, a thicker, less dense initial scavenger layer provides maximal growth. The overall conclusion is that high density clustering of members of a food chain is optimal under most common transport conditions, but under some slow transport conditions, high density clustering may not be optimal for microbial growth.
Engineering the monomer composition of polyhydroxyalkanoates synthesized in Saccharomyces cerevisiae
(2006-01) Zhang, Bo; Carlson, Ross P.; Srienc, Friedrich
Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical -oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C6 to C14) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as β-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties.
Environment Constrains Fitness Advantages of Division of Labor in Microbial Consortia Engineered for Metabolite Push or Pull Interactions
(American Society for Microbiology, 2022-08) Beck, Ashely E.; Pintar, Kathryn; Schepens, Diana; Schrammeck, Ashely; Johnson, Timothy; Bleem, Alissa; Du, Martina; Harcombe, William R.; Bernstein, Hans C.; Heys, Jeffrey J.; Gedeon, Tomas; Carlson, Ross P.
Fitness benefits from division of labor are well documented in microbial consortia, but the dependency of the benefits on environmental context is poorly understood. Two synthetic Escherichia coli consortia were built to test the relationships between exchanged organic acid, local environment, and opportunity costs of different metabolic strategies. Opportunity costs quantify benefits not realized due to selecting one phenotype over another. The consortia catabolized glucose and exchanged either acetic or lactic acid to create producer-consumer food webs. The organic acids had different inhibitory properties and different opportunity costs associated with their positions in central metabolism. The exchanged metabolites modulated different consortial dynamics. The acetic acid-exchanging (AAE) consortium had a “push” interaction motif where acetic acid was secreted faster by the producer than the consumer imported it, while the lactic acid-exchanging (LAE) consortium had a “pull” interaction motif where the consumer imported lactic acid at a comparable rate to its production. The LAE consortium outperformed wild-type (WT) batch cultures under the environmental context of weakly buffered conditions, achieving a 55% increase in biomass titer, a 51% increase in biomass per proton yield, an 86% increase in substrate conversion, and the complete elimination of by-product accumulation all relative to the WT. However, the LAE consortium had the trade-off of a 42% lower specific growth rate. The AAE consortium did not outperform the WT in any considered performance metric. Performance advantages of the LAE consortium were sensitive to environment; increasing the medium buffering capacity negated the performance advantages compared to WT.
Evaluation of cellulose as a substrate for hydrocarbon fuel production by Ascocoryne sarcoides (NRRL 50072)
(2014-02) Mallette, Natasha D.; Pankrantz, E. M.; Busse, S.; Strobel, Gary A.; Carlson, Ross P.; Peyton, Brent M.
The fungal endophyte, Ascocoryne sarcoides, produced aviation, gasoline and diesel-relevant hydrocarbons when grown on multiple substrates including cellulose as the sole carbon source. Substrate, growth stage, culturing pH, temperature and medium composition were statistically significant factors for the type and quantity of hydrocarbons produced. Gasoline range (C5-C12), aviation range (C8-C16) and diesel range (C9-C36) organics were detected in all cultured media. Numerous non-oxygenated hydrocarbons were produced such as isopentane, 3,3-dimethyl hexane and d-limonene during exponential growth phase. Growth on cellulose at 23˚C and pH 5.8 produced the highest overall yield of fuel range organics (105 mg * g·biomass−1). A change in metabolism was seen in late stationary phase from catabolism of cellulose to potential oxidation of hydrocarbons resulting in the production of more oxygenated compounds with longer carbon chain length and fewer fuel-related compounds. The results outline rational strategies for controlling the composition of the fuel-like compounds by changing culturing parameters.
Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04
(Elsevier BV, 2023-01) Goemann, Calvin L.C.; Wilkinson, Royce; Henriques, William; Bui, Huyen; Goemann, Hannah M.; Carlson, Ross P.; Viamajala, Sridhar; Gerlach, Robin; Wiedenheft, Blake
Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequence-based methods.
Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04
(Elsevier BV, 2023-01) Goemann, Calvin L.C.; Wilkinson, Royce; Henriques, William; Bui, Huyen; Goemann, Hannah M.; Carlson, Ross P.; Viamajala, Sridhar; Gerlach, Robin; Wiedenheft, Blake
Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequence-based methods.
Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desportion ionization mass spectrometry
(2012-09) Blaze, M. T.; Aydin, B.; Carlson, Ross P.; Hanley, L.
The heptapeptide ARHPHPH was identified from biofilms and planktonic cultures of two different strains of Enterococcus faecalis, V583 and ATCC 29212, using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). ARHPHPH was also imaged at the boundary of cocultured, adjacent E. faecalis and Escherichia coli (ATCC 25922) biofilms, appearing only on the E. faecalis side. ARHPHPH was proteolyzed from Îº-casein, a component in the growth media, by E. faecalis microbes. Additionally, top down and bottom up proteomic approaches were combined to identify and spatially locate multiple proteins within intact E. faecalis V583 biofilms by MALDI-MS. The resultant tandem MS data were searched against the NCBInr E. faecalis V583 database to identify thirteen cytosolic and membrane proteins which have functional association with the cell surface. Two of these proteins, enolase and GAPDH, are glycolytic enzymes known to display multiple functions in bacterial virulence in related bacterial strains. This work illustrates a powerful approach for discovering and localizing multiple peptides and proteins within intact biofilms.
In Silico Analysis of Functionalized Hydrocarbon Production Using Ehrlich Pathway and Fatty Acid Derivatives in an Endophytic Fungus
(MDPI, 2021-05) Hunt, Kristopher A.; Mallette, Natasha D.; Peyton, Brent M.; Carlson, Ross P.
Functionalized hydrocarbons have various ecological and industrial uses, from signaling molecules and antifungal/antibacterial agents to fuels and specialty chemicals. The potential to produce functionalized hydrocarbons using the cellulolytic, endophytic fungus, Ascocoryne sarcoides, was quantiﬁed using genome-enabled, stoichiometric modeling. In silico analysis identiﬁed available routes to produce these hydrocarbons, including both anabolic- and catabolic-associated strategies, and determined correlations between the type and size of the hydrocarbons and culturing conditions. The analysis quantiﬁed the limits of the wild-type metabolic network to produce functionalized hydrocarbons from cellulose-based substrates and identiﬁed metabolic engineering targets, including cellobiose phosphorylase (CP) and cytosolic pyruvate dehydrogenase complex (PDHcyt). CP and PDHcyt activity increased the theoretical production limits under anoxic conditions where less energy was extracted from the substrate. The incorporation of both engineering targets resulted in near-complete conservation of substrate electrons in functionalized hydrocarbons. The in silico framework was integrated with in vitro fungal batch growth experiments to support O2 limitation and functionalized hydrocarbon production predictions. The metabolic reconstruction of this endo-phytic ﬁlamentous fungus describes pathways for both speciﬁc and general production strategies of 161 functionalized hydrocarbons applicable to many eukaryotic hosts.
In silico approaches to study mass and energy flows in microbial consortia: A syntrophic case study
(2009) Taffs, Reed L.; Aston, John E.; Brileya, Kristen A.; Jay, Zackary J.; Klatt, Christian G.; McGlynn, Shawn E.; Inskeep, William P.; Ward, David M.; Carlson, Ross P.
Background: Three methods were developed for the application of stoichiometry-based network analysis approaches including elementary mode analysis to the study of mass and energy flows in microbial communities. Each has distinct advantages and disadvantages suitable for analyzing systems with different degrees of complexity and a priori knowledge. These approaches were tested and compared using data from the thermophilic, phototrophic mat communities from Octopus and Mushroom Springs in Yellowstone National Park (USA). The models were based on three distinct microbial guilds: oxygenic phototrophs, filamentous anoxygenic phototrophs, and sulfate-reducing bacteria. Two phases, day and night, were modeled to account for differences in the sources of mass and energy and the routes available for their exchange.ResultsThe in silico models were used to explore fundamental questions in ecology including the prediction of and explanation for measured relative abundances of primary producers in the mat, theoretical tradeoffs between overall productivity and the generation of toxic by-products, and the relative robustness of various guild interactions.Conclusion: The three modeling approaches represent a flexible toolbox for creating cellular metabolic networks to study microbial communities on scales ranging from cells to ecosystems. A comparison of the three methods highlights considerations for selecting the one most appropriate for a given microbial system. For instance, communities represented only by metagenomic data can be modeled using the pooled method which analyzes a community's total metabolic potential without attempting to partition enzymes to different organisms. Systems with extensive a priori information on microbial guilds can be represented using the compartmentalized technique, employing distinct control volumes to separate guild-appropriate enzymes and metabolites. If the complexity of a compartmentalized network creates an unacceptable computational burden, the nested analysis approach permits greater scalability at the cost of more user intervention through multiple rounds of pathway analysis. The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1752-0509/3/114