Description:

Dr. Hal Alper
Department of Chemical Engineering: The University of Texas at Austin

Engineering an Expanded Chemical Palette in Cells

An industrial biotechnology revolution is approaching. Recent technical advances are leading to a rapid transformation of the chemical palette available in cells making it conceivable to produce nearly any organic molecule of interest—from biofuels to biopolymers to pharmaceuticals. However, these feats require the ability to “hijack” native cellular machinery and metabolism and navigate the complexity inherent in cellular regulation. In this vain, this talk will describe recent advances in engineering various yeasts for the production of important products, such as organic acids and oleochemicals, with a focus on the synthetic biology tools and paradigms required along the way. Collectively, these case studies demonstrate the power and utility of using yeasts as a production host for chemicals.

Dr. Hal Alper is an Associate Professor and Fellow of the Paul D. & Betty Robertson Meek Centennial Professorship in Chemical Engineering at The University of Texas at Austin. He earned his Ph.D. in Chemical Engineering from the Massachusetts Institute of Technology in 2006 and was a postdoctoral research associate at the Whitehead Institute for Biomedical Research from 2006-2008, and at Shire Human Genetic Therapies from 2007-2008. Dr. Alper also serves on the Graduate Studies Committee for the Cell and Molecular Biology Department and the Biochemistry Department. He is currently the Principal Investigator of the Laboratory for Cellular and Metabolic Engineering at The University of Texas at Austin where his lab focuses on metabolic and cellular engineering in the context of biofuel, biochemical, and biopharmaceutical production in an array of model host organisms. His research focuses on applying and extending the approaches of related fields such as synthetic biology, systems biology, and protein engineering. Dr. Alper has published over 75 articles and 8 book chapters that have been cited over 5400 times and has an h-index of 33. Dr. Alper is the recipient of the Camille and Henry Dreyfus New Faculty Award in 2008, the Texas Exes Teaching Award in 2009, the DuPont Young Investigator Award in 2010, the Office of Naval Research Young Investigator Award in 2011, the UT Regents’ Outstanding Teaching Award in 2012, the 2013 Biotechnology and Bioengineering Daniel I.C. Wang Award, the Jay Bailey Young Investigator Award in Metabolic Engineering in 2014, the 2014 Camille Dreyfus Teacher-Scholar Award, 2015 Society for Industrial Microbiology and Biotechnology Young Investigator Award, and 2016 ACS BIOT Young Investigator Award.

Description:

Our second ChBE seminar of the Spring Quarter will be presented by two of our grad students.

Austin Isner, Ottino/Lueptow Lab
Modeling Size-based Particle Segregation in Free Surface Granular Flows

Free surface granular flows constitute a broad class of solids flow problems important to geophysics as well as industry, e.g. in the form of avalanches and deep sea lahars, or heap flows in silos and chutes. Such flows exhibit the tendency to spatially segregate based on differences in particle properties such as particle size, presenting challenges in the rational design of mixing equipment or storage bins for bulk solids. In this talk, I will present a recent continuum-based modeling approach that can be used to quantitatively predict the final segregation pattern in quasi-two-dimensional bounded heap flows of bidisperse granular mixtures consisting of two particle sizes. The successful application of the continuum theory requires detailed knowledge of the kinematics (including the mean velocity field, flowing layer thickness, diffusion coefficient, and the individual species’ segregation velocity), which can be obtained using Discrete Element Method (DEM) simulations. Scaling relations for the kinematics in terms of a wide range of physical control parameters are determined using a GPU-accelerated version of the DEM algorithm (with a 10x speed up compared to previous simulations) for simulations involving 106 particles. In the final part of my talk, I will also discuss an extension to the theory to model size segregation in multi-disperse and polydisperse systems described by a continuous particle size distribution. These extensions offer one possible approach to study the essential behavior of real segregating granular systems, with the current aim to improve our understanding of the dominant segregation mechanisms.
Funded by The Dow Chemical Company

James Jeffryes, Tyo Lab
Illuminating and Detecting Dark Metabolism

Metabolomics, the study of the population of small molecules in a cell, has drawn intense interest in fields from medicine to synthetic biology because it can provide a fine-grain representation of cellular state and activity. For this reason, metabolomics has great potential to drive the discovery of novel chemistry and pathways that have, to date, eluded discovery by other approaches. Today, the major bottleneck preventing the broader use of metabolomics data in systems biology is the identification of metabolites from their characteristic mass spectra. This process largely relies on incomplete biochemical models of the cell, which biases studies towards rediscovery of known compounds and ensures that new metabolites and pathways are seldom discovered in untargeted studies.

To address this challenge, we have constructed Metabolic In silico Network Expansions (MINEs) that expand existing metabolic models by using expert curated reaction rules to propose novel metabolites and reactions. The reaction rules include an enzymatic set, which has been demonstrated to reproduce a large fraction of known biochemical reactions and a set describing spontaneous (non-enzymatic) chemical transformations of metabolites under physiological conditions. We have applied these generalized reaction rules to compounds from various biochemical databases and resulting MINEs are freely accessible for noncommercial use at https://urldefense.proofpoint.com/v2/url?u=http-3A__minedatabase.mcs.anl&d=DwIFaQ&c=yHlS04HhBraes5BQ9ueu5zKhE7rtNXt_d012z2PA6ws&r=MYyeiQQvLWiHcXw46iyq5q34iML17wBlEqZXKlNMi3AAm7TKg3_d4AuXJrPghAqX&m=VkkagTVj84-ZFzqXvj4FzsnmxvOSvIhEuxpiIJvvZZw&s=RHM5xuUMR8NDqWHeHpH2MX2wHCzctxIySRIyQA1_xZ0&e= . The databases contain over 750,000 putative metabolites; over 90% of which are not found in PubChem, the largest freely available database of chemicals. MINEs have been used to annotate novel metabolites from 4 diverse organisms and inform our exploration of the reactions that damage labile metabolites. MINE databases shine a light on unannotated enzymatic functions and undiscovered metabolic pathways, enabling more complete and predictive models of cellular systems.

Description:

Speaker: Rey Martin, Jewett Lab

Title: Development of a CHO-based Cell-free Biomanufacturing Platform for the Synthesis of Active Monoclonal Antibodies as a Potential High-Throughput Screening Tool

Abstract
Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. At the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. Typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. I will describe our work towards developing a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid in vitro synthesis of active, aglycosylated mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of temporal addition of heavy chain and light chain plasmids for intact mAb production. Using our optimized platform, we demonstrate for the first time to our knowledge the CFPS of biologically active, intact mAb at >100 mg/L using a eukaryotic-based extract. We then also explored the utility of our system as a tool for ranking yields of candidate antibodies. Unlike stable or transient transfection-based screening, which requires a minimum of 7 days for setup and execution, results using our CHO-based CFPS platform are attained within 2 days and it is well-suited for automation. Further development would provide a tool for rapid, high throughput prediction of expression ranking of mAb producers to accelerate design-build-test cycles required for antibody expression and engineering. Looking forward, the CHO-based CFPS platform could facilitate the synthesis of toxic proteins as well.

Speaker: Quentin Dudley, Jewett Lab

Title: Bio-breadboarding: using cell-free mixing of crude extracts to prototype isoprenoid biosynthesis

Abstract
Metabolic engineering of microorganisms to produce useful compounds from renewable substrates is a promising means for sustainable, on-demand production of chemicals. However, efforts to design and engineer microbial cell factories are constrained by slow “build” times in which each genetic variation requires re-engineering a new strain for each iteration. To alleviate this challenge, we have built a plug-and-play prototyping system for isoprenoid biosynthesis. Isoprenoids are a promising class of target molecules with over 40,000 known structures and potential uses as pharmaceuticals, flavors, fragrances, pesticides, disinfectants, and chemical feedstocks. By mixing together multiple crude extracts, each enriched in a single pathway enzyme, we can recapitulate isoprenoid metabolism in a test tube which allows easy manipulation of the reaction conditions and quick testing of enzyme ratios and pathway configurations. To further minimize the time required to test enzyme homologs, we have used cell-free protein synthesis (CFPS) to synthesize pathway enzymes directly in the lysate by simply adding the appropriate DNA template along with energy molecules, cofactors, and amino acid substrate. By running nine separate CFPS reactions and mixing them together with a glucose substrate, we can generate 300 mg/L limonene (an isoprenoid fragrance molecule). This approach shortens the time from ordering genes to characterizing active enzymes to ~3 days and allows precise measurement and control of enzyme concentration. Our platform opens the possibility of extensive testing of enzymes levels and physiochemical conditions in order to prototype and accelerate in vivo metabolic engineering efforts.

Description:

Speaker: Lauren McCullough, Notestein Lab

Title: Acceptorless Dehydrogenative Coupling of Ethanol over Bulk MoS2 and Spectroscopic Structure-Function Correlations

Abstract
The production of oxygenates such as esters and organic acids from alcohols is an attractive route to high value industrial chemicals. In particular, routes that do not require the addition of oxidants, volatile or toxic reactants, base co-catalyst, or precious metal catalysts are highly desirable [1]. Acceptorless dehydrogenative coupling (ADC) has been commercialized over copper based catalysts [2] but current systems suffer from the requirement that bio-ethanol be dried before introduction to the catalyst bed and from the co-production of aldehydes and ketones requiring re-hydrogenation over a Ru based secondary catalyst. Previous reports have indicated that sulfated Mo/C could be a potentially promising material for this class of reactions [3].

In this work, we demonstrate that nanoparticulate bulk MoS2 is active for conversion of ethanol to ethyl acetate and hydrolysis to acetic acid. In high pressure batch reactions equilibrium yield of ethyl acetate (44%) is achieved in 24 hours at 230oC. Upon addition of water, total acetate yield (ethyl acetate and acetic acid) increases to 82%. MoS2 from a variety sources was tested for this reaction. Structure-function correlations were developed by combining atmospheric pressure flow reactions and material characterization based on x-ray absorption spectroscopy and infrared spectroscopy with CO as a probe molecule [4]. Based on these correlations, it is postulated that formation of ethyl acetate occurs via a hemiacetal intermediate [5] over two sites, a coordinatively unsaturated Mo site for dehydrogenation of ethanol to acetaldehyde, and a basic site (likely an -SH group) for formation of the hemiacetal [6].

This is the first report of ADC on bulk MoS2 as well as the first application of these characterization techniques to a class of reactions outside of the hydrotreating literature.

References:
(1) Sato, A. G.; Biancolli, A. L. G.; Paganin, V. A.; da Silva, G. C.; Cruz, G.; dos Santos, A. M.; Ticianelli, E. A.; Int. J. Hydrogen Energy. 2015, 40, 14716-147222.
(2) Colley, S. W.; Tuck, M. W. M.; Catalysis in Application, 2003, 101-107.
(3) Wang, L. X.; Zheng, D. F.; Ma, C. X.; Zhu, W. C.; Liu, S. Y.; Cui, J.; Wang Z, L.; Zhang, W. X.; Polish J. Chem., 2009, 83, 1993-2000.
(4) Chen, J.; Maugé, F.; El Fallah, J.; Oliviero, L.; J. Catal., 2014, 320, 170-179.
(5) Inui, K.; Kurabayashi, T.; Sato, S.; J. Catal., 2002, 380, 113-117.
(6) Colley, S. W.; Tabatabaei, J.; Waugh, K. C.; Wood, M. A.; J. Catal., 2005, 236, 21-33.

Speaker: Hongda Zhang, Snurr Lab

Title: Computational Study of Natural Gas Storage and Water Adsorption in Metal-Organic Frameworks

Abstract
Metal-Organic Frameworks (MOFs) are a new class of promising nanoporous materials for different applications including gas storage and separation, catalysis, sensor, etc. In this talk, the computational study of natural gas storage and water adsorption in MOFs will be introduced.
Adsorbed natural gas (ANG) has many advantages, including higher safety and lower cost, compared with traditional compressed natural gas storage for vehicular applications. However, in addition to methane, commercial natural gas always contains small amount of impurities including ethane and propane. These higher hydrocarbons are more easily adsorbed by the adsorbents due to their stronger interactions with the adsorbent framework atoms. In order to study the effect of these impurities on the performance of an ANG tank, we combined grand canonical Monte Carlo (GCMC) simulations and macroscopic thermodynamics to develop a model for an ANG tank. With this model, the performance, especially the deliverable energy, of the natural gas storage system with different MOFs can be tested over many operation (adsorption/desorption) cycles. Furthermore, screening of a small MOF database containing 120 structures has been carried out. Based on the screening result, good MOF candidates have been identified, and some interesting trends have been observed.
Water is one of the most common components in industrial gas or liquid systems. It often has a non-negligible effect on chemical and physical processes such as gas or liquid adsorption in porous materials, like zeolites and MOFs. However, the molecular simulation of water adsorption in MOFs always brings many challenges especially the slow simulation speed mainly due to the clustering of water molecules through hydrogen bonds. In this study we selected the hydrophobic MOF ZIF-8 as a representative adsorbent to discover the adsorption mechanism of water. In addition, we proposed and investigated several advanced Monte Carlo algorithms including the energy-bias method and the continuous fractional component Monte Carlo (CFC MC) method and successfully accelerated the simulation speed.

Description:

Title: Thermal Depolymerization of Cellulose and Lignin: The Role of Oligomeric Intermediate products.

Dr. Manuel Garcia-Perez
Washington State University

Abstract
Fast pyrolysis is a thermochemical process able to covert more than 70 wt. % of ligno-cellulosic materials into bio-oil. This oil can be further upgraded or refined for electricity, transportation fuels and chemicals production. Most of the fast pyrolysis reaction models reported in the literature suggest that monomeric products are directly produced from the depolymerization of biomass constituents (cellulose, hemicellulose and lignin) and that these monomeric molecules recombine after condensation to form oligomers. In our presentation we will show experimental evidences supporting a new description of fast pyrolysis reactions in which cellulose and lignin are first depolymerized to form a liquid intermediate of heavy oligomeric products. The acidic nature of this liquid intermediate accelerates dehydration, polycondensation and further depolymerization. These heavy molecules are removed from the liquid intermediate by micro-explosions caused by the bursting of pyrolytic vapor bubbles. In our presentation we will discuss the importance of these results to understand the underlying processes at all relevant scales, ranging from the chemistry of cell wall deconstruction to optimization of pyrolysis factories, in order to produce better quality oils for targeted uses.

Dr. Manuel Garcia-Perez is an Associate Professor in Biological Systems Engineering at WSU. For the last 15 years, he has worked on projects related to the conversion of forest and agricultural biomass into bio-fuels and chemicals, mostly via thermochemical conversion- using heat to break natural polymers into usable molecules and char. Dr. Garcia-Perez has made contributions to the understanding of thermochemical reactions of cellulose, hemicelluloses, and lignin as well as on the characterization and uses for crude bio-oils. Currently, he is working on the development of more selective pyrolysis reactors and new concepts to refine production of pyrolysis oils and engineered carbonaceous materials. Dr. Garcia-Perez is also very active in the study of ways to integrate biomass conversion technologies into existing infrastructure (e.g. pulp and paper mills, petroleum refineries, corn ethanol mills, sugar cane mills) to build bio-refineries that produce fuels and chemicals.Award in 2012, the 2013 Biotechnology and Bioengineering Daniel I.C. Wang Award, the Jay Bailey Young Investigator Award in Metabolic Engineering in 2014, the 2014 Camille Dreyfus Teacher-Scholar Award, 2015 Society for Industrial Microbiology and Biotechnology Young Investigator Award, and 2016 ACS BIOT Young Investigator Award.