Browsing by Author "Feng, Xueyang"

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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
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    Additive Manufacturing for Robust and Affordable Medical Devices
    Wolozny Gomez Robelo, Daniel Andre (Virginia Tech, 2016-10-18)
    Additive manufacturing in the form of 3D printing is a revolutionary technology that has developed within the last two decades. Its ability to print an object with accurate features down to the micro scale have made its use in medical devices and research feasible. A range of life-saving technologies can now go from the laboratory and into field with the application of 3D-printing. This technology can be applied to medical diagnosis of patients in at-risk populations. Living biosensors are limited by being Genetically Modified Organisms (GMOs) from being employed for medical diagnosis. However, by containing them within a 3D-printed enclosure, these technologies can serve as a vehicle to translate life-saving diagnosis technologies from the laboratory and into the field where the lower cost would allow more people to benefit from inexpensive diagnosis. Also, the GMO biosensors would be contained with a press-fit, ensuring that the living biosensors are unable to escape into the environment without user input. In addition, 3D-printing can also be applied to reduce the cost of lab-based technologies. Cell patterning technology is a target of interest for applying more cost-effective technologies, as elucidation of the variables defining cell patterning and motility may help explain the mechanics of cancer and other diseases. Through the use of a 3D-printed stamp, bacterial cells can be patterning without the use of a clean room, thus lowering the entry-barrier for researchers to explore cell patterning. With the commercialization of 3D-printing an opportunity has arisen to transition life-saving technologies into more cost-effective versions of existing technologies. This would not only allow more research into existing fields, but also to ensure that potentially life-saving technologies reach the people that need them.
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    C-13 Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells
    Luo, Shuai; Guo, Weihua; Nealson, Kenneth H.; Feng, Xueyang; He, Zhen (Nature Publishing Group, 2016-02-12)
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    Cell-free protein synthesis of norovirus virus-like particles
    Sheng, Jiayuan; Lei, Shaohua; Yuan, Lijuan; Feng, Xueyang (Royal Society of Chemistry, 2017-05-25)
    Norovirus vaccine development largely depends on recombinant virus-like-particles (VLPs). Norovirus VLPs have been produced in several cell-based expression systems with long production times. Here we report, for the first time, that norovirus VLPs can be expressed and assembled by using a cell-free protein expression system within four hours.
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    Computational Modeling of Planktonic and Biofilm Metabolism
    Guo, Weihua (Virginia Tech, 2017-10-16)
    Most of microorganisms are ubiquitously able to live in both planktonic and biofilm states, which can be applied to dissolve the energy and environmental issues (e.g., producing biofuels and purifying waste water), but can also lead to serious public health problems. To better harness microorganisms, plenty of studies have been implemented to investigate the metabolism of planktonic and/or biofilm cells via multi-omics approaches (e.g., transcriptomics and proteomics analysis). However, these approaches are limited to provide the direct description of intracellular metabolism (e.g., metabolic fluxes) of microorganisms. Therefore, in this study, I have applied computational modeling approaches (i.e., 13C assisted pathway and flux analysis, flux balance analysis, and machine learning) to both planktonic and biofilm cells for better understanding intracellular metabolisms and providing valuable biological insights. First, I have summarized recent advances in synergizing 13C assisted pathway and flux analysis and metabolic engineering. Second, I have applied 13C assisted pathway and flux analysis to investigate the intracellular metabolisms of planktonic and biofilm cells. Various biological insights have been elucidated, including the metabolic responses under mixed stresses in the planktonic states, the metabolic rewiring in homogenous and heterologous chemical biosynthesis, key pathways of biofilm cells for electricity generation, and mechanisms behind the electricity generation. Third, I have developed a novel platform (i.e., omFBA) to integrate multi-omics data with flux balance analysis for accurate prediction of biological insights (e.g., key flux ratios) of both planktonic and biofilm cells. Fourth, I have designed a computational tool (i.e., CRISTINES) for the advanced genome editing tool (i.e., CRISPR-dCas9 system) to facilitate the sequence designs of guide RNA for programmable control of metabolic fluxes. Lastly, I have also accomplished several outreaches in metabolic engineering. In summary, during my Ph.D. training, I have systematically applied computational modeling approaches to investigate the microbial metabolisms in both planktonic and biofilm states. The biological findings and computational tools can be utilized to guide the scientists and engineers to derive more productive microorganisms via metabolic engineering and synthetic biology. In the future, I will apply 13C assisted pathway analysis to investigate the metabolism of pathogenic biofilm cells for reducing their antibiotic resistance.
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    Controlled Hybrid Material Synthesis using Synthetic Biology
    Scott, Felicia Yi Xia (Virginia Tech, 2017-06-02)
    The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function. Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering.
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    Development of Integrated Photobioelectrochemical System (IPB): Processes, Modeling and Applications
    Luo, Shuai (Virginia Tech, 2018-04-24)
    Effective wastewater treatment is needed to reduce the water pollution problem. However, massive energy is consumed in wastewater treatment, required to design an innovative system to reduce the energy consumption to solve the energy crisis. Integrated photobioelectrochemical system (IPB) is a powerful system to combine microbial fuel cells (MFCs) and algal bioreactor together. This system has good performance on the organic degradation, removal of nitrogen and phosphorus, and recover the bioenergy via electricity generation and algal harvesting. This dissertation is divided to twelve chapters, about various aspects of the working mechanisms and actual application of IPB. Chapter 1 generally introduces the working mechanisms of MFCs, algal bioreactor, and modeling. Chapter 2 demonstrates the improvement of cathode material to improve the structure and catalytic performance to improve the MFC performance. Chapter 3 describes the process to use microbial electrolysis cell (MEC) to generate biohythane for the energy recovery. Chapters 4 and 5 demonstrate the application of stable isotope probing to study Shewanella oneidensis MR-1 in the MFCs. Chapters 6 to 8 describe the application of models to optimize MFC and IPB system performance. Chapter 9 describes the strategy improvement for the algal harvesting in IPB. Chapter 10 describes the application of scale-up bioelectrochemical systems on the long-term wastewater treatment. Chapter 11 finally concludes the perspectives of IPBs in the wastewater treatment and energy recovery. This dissertation comprehensively introduces IPB systems in the energy recovery and sustainable wastewater treatment in the future.
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    Factors that Affect the Immunogenicity of Lipid-PLGA Nanoparticle-Based Nanovaccines against Nicotine Addiction
    Zhao, Zongmin (Virginia Tech, 2017-09-06)
    Tobacco smoking has consistently been the leading cause of preventable diseases and premature deaths. Currently, pharmacological interventions have only shown limited smoking cessation efficacy and sometimes are associated with severe side effects. As an alternative, nicotine vaccines have emerged as a promising strategy to combating nicotine addiction. However, conventional conjugate nicotine vaccines have shown limited ability to induce a sufficiently strong immune response due to their intrinsic shortfalls. In this study, a lipid-poly(lactic-co-glycolic acid) (PLGA) nanoparticle-based next-generation nicotine vaccine has been developed to overcome the drawbacks of conjugate nicotine vaccines. Also, the influence of multiple factors, including nanoparticle size, hapten density, hapten localization, carrier protein, and molecular adjuvants, on its immunogenicity has been investigated. Results indicated that all these studied factors significantly affected the immunological efficacy of the nicotine nanovaccine. First, 100 nm nanovaccine was found to elicit a significantly higher anti-nicotine antibody titer than the 500 nm nanovaccine. Secondly, the high-density nanovaccine exhibited a better immunological efficacy than the low- and medium-density counterparts. Thirdly, the nanovaccine with hapten localized on both carrier protein and nanoparticle surface induced a significantly higher anti-nicotine antibody titer and had a considerably better ability to block nicotine from entering the brain of mice than the nanovaccines with hapten localized only on carrier protein or nanoparticle surface. Fourthly, the nanovaccines carrying cross reactive materials 197 (CRM197) or tetanus toxoid (TT) showed a better immunological efficacy than the nanovaccines using keyhole limpet hemocyanin (KLH) or KLH subunit as carrier proteins. Finally, the co-delivery of monophosphoryl lipid A (MPLA) and Resiquimod (R848) achieved a considerably higher antibody titer and brain nicotine reduction than only using MPLA or R848 alone as adjuvants. Collectively, the findings from this study may lead to a better understanding of the impact of multiple factors on the immunological efficacy of the hybrid nanoparticle-based nicotine nanovaccine. The findings may also provide significant guidance for the development of other drug abuse and nanoparticle-based vaccines. In addition, the optimized lipid-PLGA hybrid nanoparticle-based nicotine nanovaccine obtained by modulating the studied factors can be a promising candidate as the next-generation nicotine vaccine for treating nicotine addiction.
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    In Vitro Synthetic Biology Platform and Protein Engineering for Biorefinery
    Kim, Jae Eung (Virginia Tech, 2017-07-17)
    In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007. In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy. As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0).
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    Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via ¹³C Metabolic Flux Analysis
    Guo, Weihua; Chen, Yingying; Wei, Na; Feng, Xueyang (PLOS, 2016-08-17)
    The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, ¹³C metabolic flux analysis (¹³C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation.
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    iTAP: integrated transcriptomics and phenotype database for stress response of Escherichia coli and Saccharomyces cerevisiae
    Sundararaman, Niveda; Ash, Christine; Guo, Weihua; Button, Rebecca; Singh, Jugroop; Feng, Xueyang (2015-12-12)
    Background Organisms are subject to various stress conditions, which affect both the organism’s gene expression and phenotype. It is critical to understand microbial responses to stress conditions and uncover the underlying molecular mechanisms. To this end, it is necessary to build a database that collects transcriptomics and phenotypic data of microbes growing under various stress factors for in-depth systems biology analysis. Despite of numerous databases that collect gene expression profiles, to our best knowledge, there are few, if any, databases that collect both transcriptomics and phenotype data simultaneously. In light of this, we have developed an open source, web-based database, namely integrated transcriptomics and phenotype (iTAP) database, that records and links the transcriptomics and phenotype data for two model microorganisms, Escherichia coli and Saccharomyces cerevisiae in response to exposure of various stress conditions. Results To collect the data, we chose relevant research papers from the PubMed database containing all the necessary information for data curation including experimental conditions, transcriptomics data, and phenotype data. The transcriptomics data, including the p value and fold change, were obtained through the comparison of test strains against control strains using Gene Expression Omnibus’s GEO2R analyzer. The phenotype data, including the cell growth rate and the productivity, volumetric rate, and mass-based yield of byproducts, were calculated independently from charts or graphs within the reference papers. Since the phenotype data was never reported in a standardized format, the curation of correlated transcriptomics–phenotype datasets became extremely tedious and time-consuming. Despite the challenges, till now, we successfully correlated 57 and 143 datasets of transcriptomics and phenotype for E. coli and S. cerevisiae, respectively, and applied a regression model within the iTAP database to accurately predict over 93 and 73 % of the growth rates of E. coli and S. cerevisiae, respectively, directly from the transcriptomics data. Conclusion This is the first time that transcriptomics and phenotype data are categorized and correlated in an open-source database. This allows biologists to access the database and utilize it to predict the phenotype of microorganisms from their transcriptomics data. The iTAP database is freely available at https://sites.google.com/a/vt.edu/biomolecular-engineering-lab/software .
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    Metabolic Engineering for Production of Small Molecule Drugs: Challenges and Solutions
    Huttanus, Herbert M.; Sheng, Jiayuan; Feng, Xueyang (MDPI, 2016-02-19)
    Production of small molecule drugs in a recombinant host is becoming an increasingly popular alternative to chemical synthesis or production in natural hosts such as plants due to the ease of growing microorganisms with higher titers and less cost. While there are a wide variety of well-developed cloning techniques to produce small molecule drugs in a heterologous host, there are still many challenges towards efficient production. Therefore, this paper reviews some of these recently developed tools for metabolic engineering and categorizes them according to a chronological series of steps for a generalized method of drug production in a heterologous host, including 1) pathway discovery from a natural host, 2) pathway assembly in the recombinant host, and 3) pathway optimization to increase titers and yield.
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    Metabolic engineering of Saccharomyces cerevisiae to produce 1-hexadecanol from xylose
    Guo, Weihua; Sheng, Jiayuan; Zhao, Huimin; Feng, Xueyang (2016-02-01)
    Background An advantageous but challenging approach to overcome the limited supply of petroleum and relieve the greenhouse effect is to produce bulk chemicals from renewable materials. Fatty alcohols, with a billion-dollar global market, are important raw chemicals for detergents, emulsifiers, lubricants, and cosmetics production. Microbial production of fatty alcohols has been successfully achieved in several industrial microorganisms. However, most of the achievements were using glucose, an edible sugar, as the carbon source. To produce fatty alcohols in a renewable manner, non-edible sugars such as xylose will be a more appropriate feedstock. Results In this study, we aim to engineer a Saccharomyces cerevisiae strain that can efficiently convert xylose to fatty alcohols. To this end, we first introduced the fungal xylose utilization pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) into a fatty alcohol-producing S. cerevisiae strain (XF3) that was developed in our previous studies to achieve 1-hexadecanol production from xylose at 0.4 g/L. We next applied promoter engineering on the xylose utilization pathway to optimize the expression levels of XR, XDH, and XKS, and increased the 1-hexadecanol titer by 171 %. To further improve the xylose-based fatty alcohol production, two optimized S. cerevisiae strains from promoter engineering were evolved with the xylose as the sole carbon source. We found that the cell growth rate was improved at the expense of decreased fatty alcohol production, which indicated 1-hexadecanol was mainly produced as a non-growth associated product. Finally, through fed-batch fermentation, we successfully achieved 1-hexadecanol production at over 1.2 g/L using xylose as the sole carbon source, which represents the highest titer of xylose-based 1-hexadecanol reported in microbes to date. Conclusions A fatty alcohol-producing S. cerevisiae strain was engineered in this study to produce 1-hexadecanol from xylose. Although the xylose pathway we developed in this study could be further improved, this proof-of-concept study, for the first time to our best knowledge, demonstrated that the xylose-based fatty alcohol could be produced in S. cerevisiae with potential applications in developing consolidated bioprocessing for producing other fatty acid-derived chemicals.
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    Metabolic Engineering of Yeast to Produce Fatty Acid-derived Biofuels: Bottlenecks and Solutions
    Sheng, Jiayuan; Feng, Xueyang (Frontiers, 2015-06-08)
    Fatty acid-derived biofuels can be a better solution than bioethanol to replace petroleum fuel, since they have similar energy content and combustion properties as current transportation fuels. The environmentally friendly microbial fermentation process has been used to synthesize advanced biofuels from renewable feedstock. Due to their robustness as well as the high tolerance to fermentation inhibitors and phage contamination, yeast strains such as Saccharomyces cerevisiae and Yarrowia lipolytica have attracted tremendous attention in recent studies regarding the production of fatty acid-derived biofuels, including fatty acids, fatty acid ethyl esters, fatty alcohols, and fatty alkanes. However, the native yeast strains cannot produce fatty acids and fatty acid-derived biofuels in large quantities. To this end, we have summarized recent publications in this review on metabolic engineering of yeast strains to improve the production of fatty acid-derived biofuels, identified the bottlenecks that limit the productivity of biofuels, and categorized the appropriate approaches to overcome these obstacles.
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    Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products
    Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang (Elsevier, 2017-01-19)
    With the breakthroughs in biomolecular engineering and synthetic biology, many valuable biologically active compound and commodity chemicals have been successfully manufactured using cell-based approaches in the past decade. However, because of the high complexity of cell metabolism, the identification and optimization of rate-limiting metabolic pathways for improving the product yield is often difficult, which represents a significant and unavoidable barrier of traditional in vivo metabolic engineering. Recently, some in vitro engineering approaches were proposed as alternative strategies to solve this problem. In brief, by reconstituting a biosynthetic pathway in a cell-free environment with the supplement of cofactors and substrates, the performance of each biosynthetic pathway could be evaluated and optimized systematically. Several value-added products, including chemicals, nutraceuticals, and drug precursors, have been biosynthesized as proof-of-concept demonstrations of in vitro metabolic engineering. This mini-review summarizes the recent progresses on the emerging topic of in vitro metabolic engineering and comments on the potential application of cell-free technology to speed up the “design-build-test” cycles of biomanufacturing.
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    Molecular basis of 5-hydroxytryptophan synthesis in Saccharomyces cerevisiae
    Zhang, Jiantao; Wu, Chaochen; Sheng, Jiayuan; Feng, Xueyang (Royal Society of Chemistry, 2016-03-18)
    We report for the first time that 5-hydroxytryptophan can be synthesized in Saccharomyces cerevisiae by heterologously expressing prokaryotic phenylalanine 4-hydroxylase or eukaryotic tryptophan 3/5-hydroxylase, together with enhanced synthesis of MH4 or BH4 cofactors. The innate DFR1 gene in the folate synthesis pathway was found to play pivotal roles in 5-hydroxytryptophan synthesis.
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    OM-FBA: Integrate Transcriptomics Data with Flux Balance Analysis to Decipher the Cell Metabolism
    Guo, Weihua; Feng, Xueyang (PLOS, 2016-04-21)
    Constraint-based metabolic modeling such as flux balance analysis (FBA) has been widely used to simulate cell metabolism. Thanks to its simplicity and flexibility, numerous algorithms have been developed based on FBA and successfully predicted the phenotypes of various biological systems. However, their phenotype predictions may not always be accurate in FBA because of using the objective function that is assumed for cell metabolism. To overcome this challenge, we have developed a novel computational framework, namely omFBA, to integrate multi-omics data (e.g. transcriptomics) into FBA to obtain omics-guided objective functions with high accuracy. In general, we first collected transcriptomics data and phenotype data from published database (e.g. GEO database) for different microorganisms such as Saccharomyces cerevisiae.We then developed a “Phenotype Match” algorithm to derive an objective function for FBA that could lead to the most accurate estimation of the known phenotype (e.g. ethanol yield). The derived objective function was next correlated with the transcriptomics data via regression analysis to generate the omics-guided objective function, which was next used to accurately simulate cell metabolism at unknown conditions.We have applied omFBA in studying sugar metabolism of S. cerevisiae and found that the ethanol yield could be accurately predicted in most of the cases tested (>80%) by using transcriptomics data alone, and revealed valuable metabolic insights such as the dynamics of flux ratios. Overall, omFBA presents a novel platform to potentially integrate multi-omics data simultaneously and could be incorporated with other FBA-derived tools by replacing the arbitrary objective function with the omics-guided objective functions.
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    On-chip manufacturing of synthetic proteins for point-of-care therapeutics
    Murphy, Travis W.; Sheng, Jiayuan; Naler, Lynette B.; Feng, Xueyang; Lu, Chang (Nature, 2019)
    Therapeutic proteins have recently received increasing attention because of their clinical potential. Currently, most therapeutic proteins are produced on a large scale using various cell culture systems. However, storing and transporting these therapeutic proteins at low temperatures makes their distribution expensive and problematic, especially for applications in remote locations. To this end, an emerging solution is to use point-of-care technologies that enable immediate and accessible protein production at or near the patient’s bedside. Here we present the development of “Therapeutics-On-a-Chip (TOC)”, an integrated microfluidic platform that enables point-of-care synthesis and purification of therapeutic proteins. We used fresh and lyophilized materials for cell-free synthesis of therapeutic proteins on microfluidic chips and applied immunoprecipitation for highly efficient, on-chip protein purification. We first demonstrated this approach by expressing and purifying a reporter protein, green fluorescent protein. Next, we used TOC to produce cecropin B, an antimicrobial peptide that is widely used to control biofilmassociated diseases. We successfully synthesized and purified cecropin B at 63 ng/μl within 6 h with a 92% purity, followed by confirming its antimicrobial functionality using a growth inhibition assay. Our TOC technology provides a new platform for point-of-care production of therapeutic proteins at a clinically relevant quantity.
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    Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols
    Stevens, Joseph; Sheng, Jiayuan; Feng, Xueyang (Nature, 2016-05-27)
    Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6–C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 & C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.
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    Strategies to detoxify the mycotoxin deoxynivalenol and improve food safety in the U.S.
    Wilson, Nina Marie (Virginia Tech, 2017-06-06)
    Mycotoxins are toxic secondary metabolites produced by fungi that are a threat to the health of humans and domestic animals. The most important mycotoxin in the U.S. is deoxynivalenol (DON), which causes symptoms such as vomiting, feed refusal, and weight loss in farm animals. The fungus Fusarium graminearum produces DON in staple crops such as wheat, barley, and corn. It is estimated that the economic losses associated with DON contamination alone exceed $650 million per year in the U.S. New strategies are needed to mitigate DON and improve food safety in the U.S. The overall goal of my research is to discover and employ microorganisms and enzymes to detoxify DON. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) use a cell free protein synthesis (CFPS) system to study enzymes that modify DON, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food. In Objective 1, two mixed cultures were identified from environmental samples that converted DON into the less toxic 3-keto-deoxynivalenol (3-keto-DON). In Objective 2, a CFPS system was used to express three known acetyltransferase genes to convert DON to 3-acetyl-DON (3-A-DON). In Objective 3, we identified a potential DON transporter from a library of randomly amplified fragments from the genomes of mixed cultures of microbes isolated from the environment. In Objective 4, we developed and delivered a unique high school unit to educate high school students about potential mycotoxins in food and feed products. The work presented here represents new and improved methods for mitigating mycotoxin contamination in the United States.