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13C-Metabolic Flux Analysis: An Accurate Approach to Demystify Microbial Metabolism for Biochemical Production
Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang (MDPI, 2015-12-25)
Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to achieve a satisfactory yield, titer or productivity of the target chemicals for industrial commercialization. In order to overcome this challenge, ¹³C Metabolic Flux Analysis (¹³C-MFA) has been continuously developed and widely applied to rigorously investigate cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, many ¹³C-MFA studies have been performed in academic labs and biotechnology industries to pinpoint key issues related to microbe-based chemical production. Insightful information about the metabolic rewiring has been provided to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this review, we will introduce the basics of ¹³C-MFA and illustrate how ¹³C-MFA has been applied via integration with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production for various host microorganisms
Accelerating structure-function mapping using the ViVa webtool to mine natural variation
Hamm, Morgan; Moss, Britney; Leydon, Alexander; Gala, Hardik; Lanctot, Amy; Ramos, Román; Klaeser, Hannah; Lemmex, Andrew; Zahler, Mollye; Nemhauser, Jennifer L.; Wright, R. Clay (Wiley, 2018-12-05)
Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of this data remains inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project (http://snp-seek.irri.org/) and human genetic variation (https://www.ncbi.nlm.nih.gov/clinvar/).
Advancements in the development of HIF-1α-activated protein switches for use in enzyme prodrug therapy
Wright, R. Clay; Khakhar, Arjun; Eshleman, James R.; Ostermeier, Marc (2014-01)
While gene-directed enzyme prodrug therapy has shown potential as a cancer therapeutic in animal and clinical trials, concerns over the efficacy, selectivity, and safety of gene delivery vehicles have restricted its advance. In an attempt to relieve some of the demands on targeted gene delivery vehicles and achieve the full potential of enzyme prodrug therapy, cancer-targeted activity can be engineered into the enzyme itself. We previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells accumulating the cancer marker hypoxia-inducible factor-1α (HIF-1α). This HIF-1α-activated protein switch (Haps59) is designed to increase its ability to convert the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil in a HIF-1α-dependent manner. However, in cancer cell lines expressing Haps59 the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. In this work, we aimed to improve the cancer specificity of this switch via a directed evolution approach utilizing random mutagenesis, linker mutagenesis, and random insertion and circular permutation. We identified improved HIF-1α-activated protein switches that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. Additionally, the current bottleneck in the development of improved HIF-1α-activated protein switches is screening switch candidates in mammalian cells. To accommodate higher throughput and reduce experimental variability, we explored the use of Flp recombinase-mediated isogenic integration in 293 cells. These experiments raised the possibility that Haps59 can be activated by other interactors of the CH1 domain, and experiments in E. coli indicated that CITED2 can also activate Haps59. Although many CH1 binding partners are also oncogenes, CH1's promiscuous binding and subsequent off-target activation of Haps59 needs to be examined under normal physiological conditions to identify off-target activators. With aberrant activating molecules identified, further directed evolution can be performed to improve the cancer specificity of HIF-1α-activated protein switches.
Advances in Biochemical Engineering-Biotechnology
Zhang, Y. H. Percival; Rollin, Joseph A.; Ye, Xinhao; Del Campo, Julia S. Martin; Adams, Michael W. W. (Springer, 2014-07-15)
In vitro hydrogen generation represents a clear opportunity for novel bioreactor and system design. Hydrogen, already a globally important commodity chemical, has the potential to become the dominant transportation fuel of the future. Technologies such as in vitro synthetic pathway biotransformation (SyPaB)—the use of more than 10 purified enzymes to catalyze unnatural catabolic pathways—enable the storage of hydrogen in the form of carbohydrates. Biohydrogen production from local carbohydrate resources offers a solution to the most pressing challenges to vehicular and bioenergy uses: small-size distributed production, minimization of CO2 emissions, and potential low cost, driven by high yield and volumetric productivity. In this study, we introduce a novel bioreactor that provides the oxygen-free gas phase necessary for enzymatic hydrogen generation while regulating temperature and reactor volume. A variety of techniques are currently used for laboratory detection of biohydrogen, but the most information is provided by a continuous low-cost hydrogen sensor. Most such systems currently use electrolysis for calibration; here an alternative method, flow calibration, is introduced. This system is further demonstrated here with the conversion of glucose to hydrogen at a high rate, and the production of hydrogen from glucose 6-phosphate at a greatly increased reaction rate, 157 mmol/L/h at 60 [degrees] C.
AgroSeek: a system for computational analysis of environmental metagenomic data and associated metadata
Liang, Xiao; Akers, Kyle; Keenum, Ishi M.; Wind, Lauren L.; Gupta, Suraj; Chen, Chaoqi; Aldaihani, Reem; Pruden, Amy; Zhang, Liqing; Knowlton, Katharine F.; Xia, Kang; Heath, Lenwood S. (2021-03-10)
Background Metagenomics is gaining attention as a powerful tool for identifying how agricultural management practices influence human and animal health, especially in terms of potential to contribute to the spread of antibiotic resistance. However, the ability to compare the distribution and prevalence of antibiotic resistance genes (ARGs) across multiple studies and environments is currently impossible without a complete re-analysis of published datasets. This challenge must be addressed for metagenomics to realize its potential for helping guide effective policy and practice measures relevant to agricultural ecosystems, for example, identifying critical control points for mitigating the spread of antibiotic resistance. Results Here we introduce AgroSeek, a centralized web-based system that provides computational tools for analysis and comparison of metagenomic data sets tailored specifically to researchers and other users in the agricultural sector interested in tracking and mitigating the spread of ARGs. AgroSeek draws from rich, user-provided metagenomic data and metadata to facilitate analysis, comparison, and prediction in a user-friendly fashion. Further, AgroSeek draws from publicly-contributed data sets to provide a point of comparison and context for data analysis. To incorporate metadata into our analysis and comparison procedures, we provide flexible metadata templates, including user-customized metadata attributes to facilitate data sharing, while maintaining the metadata in a comparable fashion for the broader user community and to support large-scale comparative and predictive analysis. Conclusion AgroSeek provides an easy-to-use tool for environmental metagenomic analysis and comparison, based on both gene annotations and associated metadata, with this initial demonstration focusing on control of antibiotic resistance in agricultural ecosystems. Agroseek creates a space for metagenomic data sharing and collaboration to assist policy makers, stakeholders, and the public in decision-making. AgroSeek is publicly-available at https://agroseek.cs.vt.edu/ .
Analysis of crab meat volatiles as possible spoilage indicators for blue crab (Callinectes sapidus) meat by gas chromatography-mass spectrometry
Sarnoski, Paul J.; O'Keefe, Sean F.; Jahncke, Michael L.; Mallikarjunan, Parameswarakumar; Flick, George J. Jr. (Elsevier, 2010-10-01)
Traditionally crab meat spoilage has been evaluated using sensory panels. A method was developed using solid-phase microextraction–gas chromatography–mass spectrometry (SPME–GC–MS) to examine the aroma profile of blue crab (Callinectes sapidus) for chemical indicators of spoilage. The chemicals found to correlate best with spoilage were trimethylamine (TMA), ammonia, and indole over a period of 7 days. In addition, chemicals previously not identified in the aroma profile of blue crab were tentatively detected. Scan mode of the mass spectrometer was used to qualitatively determine compounds extracted from the volatile profile of spoiling blue crab by the SPME fiber. Selected ion monitoring (SIM) mode of the mass spectrometer improved resolution, identified compounds at low concentrations, and allowed spoilage related compounds to be detected in one chromatographic run without sample heating. TMA increased linearly. A significant difference in TMA concentrations were found for day 0 and day 4 samples. Indole concentrations corresponded well with sensory and microbial evaluations, in early, mid, and highly spoiled crab meat samples.
Armored kinorhynch-like scalidophoran animals from the early Cambrian
Zhang, H.; Xiao, S.; Liu, Y.; Yuan, X.; Wan, B.; Muscente, A. D.; Shao, T.; Gong, H.; Cao, G. (2015-11-26)
Morphology-based phylogenetic analyses support the monophyly of the Scalidophora (Kinorhyncha, Loricifera, Priapulida) and Nematoida (Nematoda, Nematomorpha), together constituting the monophyletic Cycloneuralia that is the sister group of the Panarthropoda. Kinorhynchs are unique among living cycloneuralians in having a segmented body with repeated cuticular plates, longitudinal muscles, dorsoventral muscles, and ganglia. Molecular clock estimates suggest that kinorhynchs may have diverged in the Ediacaran Period. Remarkably, no kinorhynch fossils have been discovered, in sharp contrast to priapulids and loriciferans that are represented by numerous Cambrian fossils. Here we describe several early Cambrian (~535 million years old) kinorhynch-like fossils, including the new species Eokinorhynchus rarus and two unnamed but related forms. E. rarus has characteristic scalidophoran features, including an introvert with pentaradially arranged hollow scalids. Its trunk bears at least 20 annuli each consisting of numerous small rectangular plates, and is armored with five pairs of large and bilaterally placed sclerites. Its trunk annuli are reminiscent of the epidermis segments of kinorhynchs. A phylogenetic analysis resolves E. rarus as a stem-group kinorhynch. Thus, the fossil record confirms that all three scalidophoran phyla diverged no later than the Cambrian Period.
Artificial sinks to treat legacy nutrients in agricultural landscapes
Bock, Emily; Stephenson, Stephen Kurt; Easton, Zachary M. (2019-06-05)
Legacy nutrients introduce a critical time lag between changes in nutrient application or implementation of best management practices (BMPs) and observable reductions in loads delivered to downstream waters. Nitrogen and phosphorus leached through soils into groundwater may take decades to eventually be discharged to surface waters and, consequently, often prevent the attainment of water quality improvement goals. For example, the National Resource Council has cautioned that in the Chesapeake Bay watershed legacy nutrients, particularly nitrogen (N), could delay achievement of nutrient load reductions needed to meet Total Maximum Daily Load (TMDL) requirements.. Groundwater discharge transporting legacy N has been identified specifically as a significant nutrient source to the Bay. Unfortunately, most existing BMPs cannot remediate these nutrient reservoirs and the Chesapeake Bay Program has not active policy to address legacy nutrients; better management options are needed...
Assembly of Bio-Nanoparticles for Double Controlled Drug Release
Huang, Wei; Zhang, Jianfei; Dorn, Harry C.; Zhang, Chenming (PLOS, 2013-09-06)
A critical limiting factor of chemotherapy is the unacceptably high toxicity. The use of nanoparticle based drug carriers has significantly reduced the side effects and facilitated the delivery of drugs. Source of the remaining side effect includes (1) the broad final in vivo distribution of the administrated nanoparticles, and (2) strong basal drug release from nanoparticles before they could reach the tumor. Despite the advances in pH-triggered release, undesirable basal drug release has been a constant challenge under in vivo conditions. In this study, functionalized single walled carbon nanohorn supported immunoliposomes were assembled for paclitaxel delivery. The immunoliposomes were formulated with polyethylene glycol, thermal stable and pH sensitive phospholipids. Each nanohorn was found to be encapsulated within one immunoliposome. Results showed a highly pH dependent release of paclitaxel in the presence of serum at body temperature with minimal basal release under physiological conditions. Upon acidification, paclitaxel was released at a steady rate over 30 days with a cumulative release of 90% of the loaded drug. The drug release results proved our hypothesized double controlled release mechanism from the nanoparticles. Other results showed the nanoparticles have doubled loading capacity compared to that of traditional liposomes and higher affinity to breast cancer cells overexpressing Her2 receptors. Internalized nanoparticles were found in lysosomes.
Assessing Strontium and Vulnerability to Strontium in Private Drinking Water Systems in Virginia
Scott, Veronica; Juran, Luke; Ling, Erin; Benham, Brian L.; Spiller, Asa (MDPI, 2020-04-08)
A total of 1.7 million Virginians rely on private drinking water (PDW) systems and 1.3 million of those people do not know their water quality. Because most Virginians who use PDW do not know the quality of that water and since strontium poses a public health risk, this study investigates sources of strontium in PDW in Virginia and identifies the areas and populations most vulnerable. Physical factors such as rock type, rock age, and fertilizer use have been linked to elevated strontium concentrations in drinking water. Social factors such as poverty, poor diet, and adolescence also increase social vulnerability to health impacts of strontium. Using water quality data from the Virginia Household Water Quality Program (VAHWQP) and statistical and spatial analyses, physical vulnerability was found to be highest in the Ridge and Valley province of Virginia where agricultural land use and geologic formations with high strontium concentrations (e.g., limestone, dolomite, sandstone, shale) are the dominant aquifer rocks. In terms of social vulnerability, households with high levels of strontium are more likely than the average VAHWQP participant to live in a food desert. This study provides information to help 1.7 million residents of Virginia, as well as populations in neighboring states, understand their risk of exposure to strontium in PDW.
Assessing the Effects of Climate Change on Water Quantity and Quality in an Urban Watershed Using a Calibrated Stormwater Model
Alamdari, Nasrin; Sample, David J.; Steinberg, Peter; Ross, Andrew C.; Easton, Zachary M. (MDPI, 2017-06-27)
Assessing climate change (CC) impacts on urban watersheds is difficult due to differences in model spatial and temporal scales, making prediction of hydrologic restoration a challenge. A methodology was developed using an autocalibration tool to calibrate a previously developed Storm Water Management Model (SWMM) of Difficult Run in Fairfax, Virginia. Calibration was assisted by use of multi-objective optimization. Results showed a good agreement between simulated and observed data. Simulations of CC for the 2041–2068 period were developed using dynamically downscaled North American Regional CC Assessment Program models. Washoff loads were used to simulate water quality, and a method was developed to estimate treatment performed in stormwater control measures (SCMs) to assess water quality impacts from CC. CC simulations indicated that annual runoff volume would increase by 6.5%, while total suspended solids, total nitrogen, and total phosphorus would increase by 7.6%, 7.1%, and 8.1%, respectively. The simulations also indicated that within season variability would increase by a larger percentage. Treatment practices (e.g., bioswale) that were intended to mitigate the negative effects of urban development will need to deal with additional runoff volumes and nutrient loads from CC to achieve the required water quality goals.
Automatic Calibration Tool for Hydrologic Simulation Program-FORTRAN Using a Shuffled Complex Evolution Algorithm
Seong, Chounghyun; Her, Younggu; Benham, Brian L. (MDPI, 2015-02-04)
Hydrologic Simulation Program-Fortran (HSPF) model calibration is typically done manually due to the lack of an automated calibration tool as well as the difficulty of balancing objective functions to be considered. This paper discusses the development and demonstration of an automated calibration tool for HSPF (HSPF-SCE). HSPF-SCE was developed using the open source software “R”. The tool employs the Shuffled Complex Evolution optimization algorithm (SCE-UA) to produce a pool of qualified calibration parameter sets from which the modeler chooses a single set of calibrated parameters. Six calibration criteria specified in the Expert System for the Calibration of HSPF (HSPEXP) decision support tool were combined to develop a single, composite objective function for HSPF-SCE. The HSPF-SCE tool was demonstrated, and automated and manually calibrated model performance were compared using three Virginia watersheds, where HSPF models had been previously prepared for bacteria total daily maximum load (TMDL) development. The example applications demonstrate that HSPF-SCE can be an effective tool for calibrating HSPF.
A Bayesian Assignment Method for Ambiguous Bisulfite Short Reads
Tran, H.; Wu, X.; Tithi, S.; Sun, M.-A.; Xie, H.; Zhang, L. (PLOS, 2016-03-24)
DNA methylation is an epigenetic modification critical for normal development and diseases. The determination of genome-wide DNA methylation at single-nucleotide resolution is made possible by sequencing bisulfite treated DNA with next generation high-throughput sequencing. However, aligning bisulfite short reads to a reference genome remains challenging as only a limited proportion of them (around 50–70%) can be aligned uniquely; a significant proportion, known as multireads, are mapped to multiple locations and thus discarded from downstream analyses, causing financial waste and biased methylation inference. To address this issue, we develop a Bayesian model that assigns multireads to their most likely locations based on the posterior probability derived from information hidden in uniquely aligned reads. Analyses of both simulated data and real hairpin bisulfite sequencing data show that our method can effectively assign approximately 70% of the multireads to their best locations with up to 90% accuracy, leading to a significant increase in the overall mapping efficiency. Moreover, the assignment model shows robust performance with low coverage depth, making it particularly attractive considering the prohibitive cost of bisulfite sequencing. Additionally, results show that longer reads help improve the performance of the assignment model. The assignment model is also robust to varying degrees of methylation and varying sequencing error rates. Finally, incorporating prior knowledge on mutation rate and context specific methylation level into the assignment model increases inference accuracy. The assignment model is implemented in the BAM-ABS package and freely available at https://github.com/zhanglabvt/BAM_ABS.
Biomanufacturing by in vitro biosystems containing cascade enzymes
Zhang, Y. P.; You, C. (2016)
Cell-free protein synthesis energized by slowly-metabolized maltodextrin
Wang, Yiran; Zhang, Y. H. Percival (2009-06-28)
Background Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS. Results Distinct from common secondary energy sources (e.g., phosphoenolpyruvate - PEP, glucose-6-phosphate), maltodextrin was used for energizing CFPS through substrate phosphorylation and the glycolytic pathway because (i) maltodextrin can be slowly catabolized by maltodextrin phosphorylase for continuous ATP regeneration, (ii) maltodextrin phosphorylation can recycle one phosphate per reaction for glucose-1-phosphate generation, and (iii) the maltodextrin chain-shortening reaction can produce one ATP per glucose equivalent more than glucose can. Three model proteins, esterase 2 from Alicyclobacillus acidocaldarius, green fluorescent protein, and xylose reductase from Neurospora crassa were synthesized for demonstration. Conclusion Slowly-metabolized maltodextrin as a low-cost secondary energy compound for CFPS produced higher levels of proteins than PEP, glucose, and glucose-6-phospahte. The enhancement of protein synthesis was largely attributed to better-controlled phosphate levels (recycling of inorganic phosphate) and a more homeostatic reaction environment.
Changes in Fluvial Erosion of Cohesive Streambank Soils with Stream Chemistry
Thompson, Theresa M.; Akinola, Akinrotimi I.; Hoomehr, Siavash; Eick, Matthew J. (2018-06-01)
The goal of this study was to quantify changes in fluvial erosion rates with changes in stream chemistry.
Characterizing Auxin Response Circuits in Saccharomyces cerevisiae by Flow Cytometry
Pierre-Jerome, Edith; Wright, R. Clay; Nemhauser, Jennifer L. (2017-01)
Recapitulation of the nuclear auxin response pathway in Saccharomyces cerevisiae (yeast) provides a means to functionally assay the contribution of individual signaling components to response dynamics. Here, we describe a time course assay for characterizing auxin response circuits using flow cytometry. This method allows for quantitative measurements of the dynamic response of up to 12 circuits (strains) at once. We also describe a steady-state assay and how to utilize an R package we developed to facilitate data analysis.
Characterizing the Phenotypic Responses of Escherichia coli to Multiple 4-Carbon Alcohols with Raman Spectroscopy
Zu, Theresah N. K.; Athamneh, Ahmad I. M.; Senger, Ryan S. (MDPI, 2016-01-25)
The phenotypic responses of E. coli cells exposed to 1.2% (v/v) of 1-butanol, 2-butanol, isobutanol, tert-butanol, and 1,4-butanediol were studied in near real-time using Raman spectroscopy. A method of “chemometric fingerprinting” was employed that uses multivariate statistics (principal component analysis and linear discriminant analysis) to identify E. coli phenotypic changes over a 180 min post-treatment time-course. A toxicity study showed extreme variability among the reduction in culture growth, with 1-butanol showing the greatest toxicity and 1,4-butanediol showing relatively no toxicity. Chemometric fingerprinting showed distinct phenotype clusters according to the type of alcohol: (i) 1-butanol and 2-butanol (straight chain alcohols); (ii) isobutanol and tert-butanol (branched chain alcohols); and (iii) control and 1,4-butanediol (no terminal alkyl end) treated cells. While the isobutanol and tert-butanol treated cells led to similar phenotypic responses, isobutanol was significantly more toxic. In addition, the phenotypic response was found to take place largely within 60 min of culture treatment; however, significant responses (especially for 1,4-butanediol) were still occurring at 180 min post-treatment. The methodology presented here identified different phenotypic responses to seemingly similar 4-carbon alcohols and can be used to study phenotypic responses of virtually any cell type under any set of environmental conditions or genetic manipulations.
Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(+) with Its Application to Biobatteries
Chen, Hui; Zhu, Zhiguang; Huang, Rui; Zhang, Y. H. Percival (Nature Publishing Group, 2016-11-02)
Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP(+) to NAD(+). Through amino acid-sequence alignment of NADP(+)-and NAD(+)-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP(+) were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a x 6.4 x 10(4)-fold reversal of the coenzyme selectivity from NADP(+) to NAD(+). The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm(-2) and 0.255 mA cm(-2), similar to 25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 degrees C, leading to a high power density of 1.75 mW cm(-2). This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.
Comparison of kinetic models to describe high pressure and gamma irradiation used to inactivate Vibrio vulnificus and Vibrio parahaemolyticus prepared in buffer solution and whole oysters
Hu, X.; Mallikarjunan, Kumar; Koo, J.; Jahncke, M. L. (2005-03-01)

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