preparation-washed-human-platelets-for-quantitative-metabolic-flux

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated ...

Colorado School of Mines

Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible.

Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

Genome engineering of cyanobacteria is a promising area of development in order to produce fuels, feedstocks, and value-added chemicals in a sustainable way. Unfortunately, the current state of genome engineering tools for cyanobacteria lags far behind those of model organisms such as Escherichia coli and Saccharomyces cerevisiae. In this review, we present the current state of synthetic biology tools for genome engineering efforts in the most widely used cyanobacteria strains and areas that need concerted research efforts to improve tool development. Cyanobacteria pose unique challenges to genome engineering efforts because their cellular biology differs significantly from other eubacteria; therefore, tools developed for other genera are not directly transferrable. Standardized parts, such as promoters and ribosome binding sites, which control gene expression, require characterization in cyanobacteria in order to have fully predictable results. The application of these tools to genome engineering efforts is also discussed; the ability to do genome-wide searching and to introduce multiple mutations simultaneously is an area that needs additional research in order to enable fast and efficient strain engineering.

Genome Engineering in Cyanobacteria: Where We Are and Where We Need To Go.

Special Issue on Genome Engineering.

In response to demands for sustainable domestic fuel sources, research into biofuels has become increasingly important. Many challenges face biofuels in their effort to replace petroleum fuels, but rational strain engineering of algae and photosynthetic organisms offers a great deal of promise. For decades, mutations and stress responses in photosynthetic microbiota were seen to result in production of exciting high-energy fuel molecules, giving hope but minor capability for design. However, '-omics' techniques for visualizing entire cell processing has clarified biosynthesis and regulatory networks. Investigation into the promising production behaviors of the model organism C. reinhardtii and its mutants with these powerful techniques has improved predictability and understanding of the diverse, complex interactions within photosynthetic organisms. This new equipment has created an exciting new frontier for high-throughput, predictable engineering of photosynthetically produced carbon-neutral biofuels.

Omics in Chlamydomonas for Biofuel Production.

Computational, genome based predictions of organism phenotypes has enhanced the ability to investigate the biological phenomena that help organisms survive and respond to their environments. In this study, we have created the first genome-scale metabolic network reconstruction of the nitrogen fixing cyanobacterium T. erythraeum and used genome-scale modeling approaches to investigate carbon and nitrogen fluxes as well as growth and equilibrium population composition.

The use of genome-scale metabolic network reconstruction to predict fluxes and equilibrium composition of N-fixing versus C-fixing cells in a diazotrophic cyanobacterium, Trichodesmium erythraeum.

Photosynthetic microorganisms have the potential for sustainable production of chemical feedstocks and products but have had limited success due to a lack of tools and deeper understanding of metabolic pathway regulation. The application of instationary metabolic flux analysis (INST-MFA) to photosynthetic microorganisms has allowed researchers to quantify fluxes and identify bottlenecks and metabolic inefficiencies to improve strain performance or gain insight into cellular physiology. Additionally, flux measurements can also highlight deviations between measured and predicted fluxes, revealing weaknesses in metabolic models and highlighting areas where a lack of understanding still exists. In this review, we outline the experimental steps necessary to successfully perform photosynthetic flux experiments and analysis. We also discuss the challenges unique to photosynthetic microorganisms and how to account for them, including: light supply, quenching, concentration, extraction, analysis, and flux calculation. We hope that this will enable a larger number of researchers to successfully apply isotope assisted metabolic flux analysis (C-MFA) to their favorite photosynthetic organism.

The challenge and potential of photosynthesis: unique considerations for metabolic flux measurements in photosynthetic microorganisms.

In natural environments, cells live in complex communities and experience a high degree of heterogeneity internally and in the environment. Even in 'ideal' laboratory environments, cells can experience a high degree of heterogeneity in their environments. Unfortunately, most of the metabolic modeling approaches that are currently used assume ideal conditions and that each cell is identical, limiting their application to pure cultures in well-mixed vessels. Here we describe our development of Multiscale Multiobjective Systems Analysis (MiMoSA), a metabolic modeling approach that can track individual cells in both space and time, track the diffusion of nutrients and light and the interaction of cells with each other and the environment. As a proof-of concept study, we used MiMoSA to model the growth of Trichodesmium erythraeum, a filamentous diazotrophic cyanobacterium which has cells with two distinct metabolic modes. The use of MiMoSA significantly improves our ability to predictively model metabolic changes and phenotype in more complex cell cultures.

Multiscale Multiobjective Systems Analysis (MiMoSA): an advanced metabolic modeling framework for complex systems.

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002.

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.

Isotopically nonstationary C metabolic flux analysis in resting and activated human platelets.

Increasing concentrations of atmospheric CO are leading to rising global temperatures and extreme weather events. However, the most prominent method of removing CO via direct air capture remains cost-prohibitive. Oceans sequester carbon through several naturally occurring carbon dioxide removal (CDR) processes, one of which includes microorganisms that utilize dissolved inorganic carbon (DIC) in their metabolic processes. Atmospheric CO is in dynamic equilibrium with DIC at the ocean's surface. Thus, ocean-based CDR can function to capture carbon from the air indirectly. This work discusses a hybrid method that combines primary CO capture via the growth of autotrophic microorganisms (i.e., photosynthetic cyanobacteria) and microbially induced carbonate precipitation. Carbon fixation and carbonate precipitation can be co-optimized using bipolar membrane electrodialysis (BPMED) devices , which generate seawater with an adjustable pH. We examine the scale-up potential for naturally produced bio-carbonate composite material and compare its production with published ocean CDR strategies for reducing anthropogenic CO emissions.

Biocement from the ocean: Hybrid microbial-electrochemical mineralization of CO.

The open ocean is an extremely competitive environment, partially due to the dearth of nutrients. Trichodesmium erythraeum, a marine diazotrophic cyanobacterium, is a keystone species in the ocean due to its ability to fix nitrogen and leak 30 to 50% into the surrounding environment, providing a valuable source of a necessary macronutrient to other species. While there are other diazotrophic cyanobacteria that play an important role in the marine nitrogen cycle,  is unique in its ability to fix both carbon and nitrogen simultaneously during the day without the use of specialized cells called heterocysts to protect nitrogenase from oxygen. Here, we use the advanced modeling framework called ultscale ultibjective ystems nalysis (MiMoSA) to investigate how Trichodesmium erythraeum can reduce dimolecular nitrogen to ammonium in the presence of oxygen. Our simulations indicate that nitrogenase inhibition is best modeled as Michealis-Menten competitive inhibition and that cells along the filament maintain microaerobia using high flux through Mehler reactions in order to protect nitrogenase from oxygen. We also examined the effect of location on metabolic flux and found that cells at the end of filaments operate in distinctly different metabolic modes than internal cells despite both operating in a photoautotrophic mode. These results give us important insight into how this species is able to operate photosynthesis and nitrogen fixation simultaneously, giving it a distinct advantage over other diazotrophic cyanobacteria because they can harvest light directly to fuel the energy demand of nitrogen fixation.  Trichodesmium erythraeum is a marine cyanobacterium responsible for approximately half of all biologically fixed nitrogen, making it an integral part of the global nitrogen cycle. Interestingly, unlike other nitrogen-fixing cyanobacteria,  does not use temporal or spatial separation to protect nitrogenase from oxygen poisoning; instead, it operates photosynthesis and nitrogen fixation reactions simultaneously during the day. Unfortunately, the exact mechanism the cells utilize to operate carbon and nitrogen fixation simultaneously is unknown. Here, we use an advanced metabolic modeling framework to investigate and identify the most likely mechanisms  uses to protect nitrogenase from oxygen. The model predicts that cells operate in a microaerobic mode, using both respiratory and Mehler reactions to dramatically reduce intracellular oxygen concentrations.

Investigating the Unique Ability of  To Fix Carbon and Nitrogen Simultaneously Using MiMoSA.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

The cultivated meat industry, also known as cell-based meat, cultured meat, lab-grown meat, or meat alternatives, is a growing field that aims to generate animal tissues ex-vivo in a cost-effective manner that achieves price parity with traditional agricultural products. However, cell culture media costs account for 55%-90% of production costs. To address this issue, efforts are aimed at optimizing media composition. Systems biology-driven approaches have been successfully used to improve the biomass and productivity of multiple bioproduction platforms, like Chinese hamster ovary cells, by accelerating the development of cell line-specific media and reducing research and development and production costs related to cell media and its optimization. In this review, we summarize systems biology modeling approaches, methods for cell culture media and bioprocess optimization, and metabolic studies done in animals of interest to the cultivated meat industry. More importantly, we identify current gaps in knowledge that prevent the identification of metabolic bottlenecks. These include the lack of genome-scale metabolic models for some species (pigs and ducks), a lack of accurate biomass composition studies for different growth conditions, and  C-metabolic flux analysis (MFA) studies for many of the species of interest for the cultivated meat industry (only shrimp and duck cells have been subjected to  C-MFA). We also highlight the importance of characterizing the metabolic requirements of cells at the organism, breed, and cell line-specific levels, and we outline future steps that this nascent field needs to take to achieve price parity and production efficiency similar to those of other bioproduction platforms. Practical Application: Our article summarizes systems biology techniques for cell culture media design and bioprocess optimization, which may be used to significantly reduce cell-based meat production costs. We also present the results of experimental studies done on some of the species of interest to the cultivated meat industry and highlight why modeling approaches are required for multiple species, cell-types, and cell lines.

Systems biology and metabolic modeling for cultivated meat: A promising approach for cell culture media optimization and cost reduction.

One of the main challenges with using flat panel photobioreactors for algal growth is uneven mixing and settling of cells in corners, especially when bubbling is the only method used for mixing. In order to improve mixing in our flat panel reactor, we designed a custom paddlewheel. Paddlewheels are frequently used in outdoor algae raceway ponds to improve mixing and we are taking advantage of the same principle for mixing in the reactor. The paddlewheel is easily integrated into our PSI FMT150 1-L flat panel photobioreactor and is printed on a 3D printer using high temperature poly lactic acid (HT-PLA). With the inclusion of an annealing step, the paddlewheel is autoclavable. Addition of the paddlewheel in the reactor minimized cell settling and improved algal growth, as evidenced by a nearly 40% increase in oxygen production rates. Nutrient dispersion and utilization in the culture was also improved as evidenced by a corresponding 38% decrease in CO concentration. The paddlewheel device presented here is a cost-effective method for improving algal growth in a flat panel photobioreactor.

Article	Year
Multiplexed tracking of combinatorial genomic mutations in engineered cell populations. Nature biotechnology\| PubMed ID: 25798935	2015
Genome Engineering in Cyanobacteria: Where We Are and Where We Need To Go. ACS synthetic biology\| PubMed ID: 25985322	2015
Special Issue on Genome Engineering. ACS synthetic biology\| PubMed ID: 26585869	2015
Omics in Chlamydomonas for Biofuel Production. Sub-cellular biochemistry\| PubMed ID: 27023246	2016
The use of genome-scale metabolic network reconstruction to predict fluxes and equilibrium composition of N-fixing versus C-fixing cells in a diazotrophic cyanobacterium, Trichodesmium eryth... BMC systems biology\| PubMed ID: 28103880	2017
The challenge and potential of photosynthesis: unique considerations for metabolic flux measurements in photosynthetic microorganisms. Biotechnology letters\| PubMed ID: 30430405	2019
Multiscale Multiobjective Systems Analysis (MiMoSA): an advanced metabolic modeling framework for complex systems. Scientific reports\| PubMed ID: 31740694	2019
Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002. Biotechnology progress\| PubMed ID: 32388924	2020
Isotopically nonstationary C metabolic flux analysis in resting and activated human platelets. Metabolic engineering\| PubMed ID: 34954086	2022
Biocement from the ocean: Hybrid microbial-electrochemical mineralization of CO. iScience\| PubMed ID: 36212025	2022
Investigating the Unique Ability of To Fix Carbon and Nitrogen Simultaneously Using MiMoSA. mSystems\| PubMed ID: 36598239	2023
Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive. The Plant cell\| PubMed ID: 36970811	2023
Systems biology and metabolic modeling for cultivated meat: A promising approach for cell culture media optimization and cost reduction. Comprehensive reviews in food science and food safety\| PubMed ID: 37306528	2023
A custom 3D printed paddlewheel improves growth in flat panel photobioreactor. Biotechnology progress\| PubMed ID: 38247163	2024

Nanette Boyle

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