ChBE's third seminar of the Spring Quarter will be presented by three ChBE graduate students, detailed information is given below:
Date & Time: Thursday, April 14th 8:30 am – 10:00 am
Location: Ford ITW Room (refreshments will be available at 8:25am)
Speaker: Yuan Yao, PhD candidate in the Masanet Lab
Title: Prospective Life-cycle Technology Assessment Modeling Framework for Sustainable Chemical Production
Abstract
Assessing the energy, environmental, and economic impacts of emerging technologies is critical for promoting sustainable chemical production because it provides policy makers with good references on future investment and technology selection, it also provides manufacturers and researchers with great understandings of technology potential, possible bottlenecks, and future RD&D directions. However, the assessment of new technologies is challenging for the lack of process data, general evaluation approaches across different products, and robust methodologies over the large temporal and spatial scales. This talk will present a novel technology assessment modeling framework that systematically integrates engineering, economic, and environmental life-cycle analysis approaches. Applications of this modeling framework will be discussed to demonstrate the functionality. Examples of how this modeling framework quantifies the net energy, emissions, and economic implications of pathway/technology changes for U.S. chemicals production systems and evaluates their effects across the economy from a life cycle perspective will be presented.
Speaker: Jennifer Kay, PhD Candidate in the Jewett Lab
Title: Conversion of glucose to 2,3-butanediol in Escherichia coli cell-free lysates: a model system
Abstract
In recent decades we are seeing more and more examples of whole pathways being activated in crude lysates to generate a desired product in vitro. For instance, inexpensive substrates glucose or maltose have been used to regenerate ATP to power cell-free protein synthesis. Buffers and reaction conditions designed to mimic the cytoplasm have allowed a remarkable number of enzymes to be coordinately activated and to remain stable for up to about a week. It begs the question, of whether cytoplasmic mimicry will benefit other endeavors such as rapid enzyme screening or in vitro multi-enzyme catalysis.
Here, as a model study, we sought to determine if the conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications, could be achieved in an in vitro system designed to have high cofactor regeneration. Extracts of Escherichia coli expressing three heterologous enzymes are able to convert glucose to m2,3-BD at surprisingly high rates and concentrations. With no strain optimization, we have observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD / g glucose) and concentrations of 82 ± 8 g/L m2,3-BD in batch reactions. We have found the system to be robust to working concentrations of antibiotics and antifoam, and other compounds that are toxic to cell growth but do not denature or inhibit relevant enzymes. Current work includes tuning enzyme ratios to try to understand what is necessary and limiting in the system, and testing potential enzyme screening protocols. These results highlight the ability for high-level co-factor regeneration in cell-free lysates and suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation.
Speaker: Jennifer Schoborg, PhD candidate in the Jewett Lab
Title: Rapid production of membrane-bound oligosaccharyltransferases with the aid of cell-free protein synthesis
Abstract
Protein glycosylation is the addition of sugars, called glycans, to proteins. This process is critical for pharmaceutical protein stability, activity, and immunogenicity, yet our understanding of the functional and structural consequences of site-specific glycosylation has remained limited. In order to learn more about this process, the key enzymes must be synthesized and characterized. Oligosaccharyltransferases (OSTs), which facilitate the attachment of glycans to protein targets, have historically been difficult enzymes to produce, due to their large size (80-100 kDa) and multiple transmembrane passes (approximately 12). These qualities make OSTs toxic to produce in large quantities in cells, but perfect candidates for cell-free protein synthesis (CFPS). Here, I show that CFPS allows for a novel production pipeline for rapid synthesis of a variety of active OSTs, without the need for complex cellular growth schemes. Through optimization of the CFPS reaction, we have been able to produce up to 860 mg/l of six bacterial OSTs and two eukaryotic OSTs. Additionally, four of the bacterial OSTs show activity in in vitro glycosylation reactions. We anticipate this broadly applicable production method will contribute to improved capabilities for controllable glycosylation. It will also provide new avenues to improve our knowledge of glycobiology, making possible a new era of applications in glycoprotein therapeutics and glycoconjugate vaccines.