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BIOT 1 Maturation of Stem Cell-Derived Skeletal Myocytes DRAFT BIOT 1 Maturation of stem cell-derived skeletal myocytes promoted by micropatterning and substrate stiffness Wendy Crone, Nunnapas Jiwlawat, Brett Napiwocki, Eileen Lynch, Alana Stempien, Randy Ashton, Tim Kamp, Masatoshi Suzuki, [email protected]. University oF Wisconsin-Madison, Madison, Wisconsin, United States In this work we demonstrate the effectiveness of an engineered two- dimensional micropatterned cell culture platform for creating highly aligned myotubes from human induced pluripotent stem cell (iPSC)-derived myogenic progenitors. Myotube elongation was shown to be dependent on the micropattern feature width and spontaneous contractions were aligned with the long axis of the pattern. As a result of an optimized micropattern feature geometry on a substrate of physiologically relevant stiffness, the resulting myotubes were elongated, well-aligned, and similar to myofibers; they showed significant improvement in nuclear alignment, myotube fusion, and sarcomere formation. This platform holds great potential in further understanding the process of human muscle development, as well as opportunities for in vitro pharmacological studies with skeletal muscle diseases. As an example, we demonstrated that bundle-like myotubes can be produced using patient- derived iPSCs with a background of Pompe disease (glycogen storage disease type II). Culturing in this engineered platform enhanced the disease phenotype as demonstrated by the observation of abnormal lysosome accumulations. BIOT 2 Cell free platform for rapid synthesis and testing of active oligosaccharyltransferases Jasmine M. Hershewe, [email protected], Jennifer Schoborg, Michael C. Jewett. Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, United States Protein glycosylation, the covalent attachment of sugar moieties to proteins, is important for protein stability, activity, and immunogenicity. Recombinant glycoproteins are critical in biotechnology, comprising life-saving therapeutics and vaccines. However, despite its importance, biomanufacturing defined glycoproteins and understanding the structure/ function relationships of DRAFT glycosylation remains a significant challenge due to technological limitations. These limitations include a lack of available tools for high-throughput biochemical characterization of enzymes that carry out glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of complex, pre-built glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large and contain many (>10) transmembrane helices. Here, we address this challenge by establishing a bacterial cell free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained soluble yields of up to 420 mg/L for the single subunit OST, PglB, from Campylobacter jejuni, as well as for three additional bacterial PglB homologs. Importantly, the cell free derived enzymes catalyzed glycosylation reactions in vitro with no purification or processing needed, and the ability to tightly control concentrations and ratios of glycosylation components in the in vitro system allowed us to quickly optimize for full glycosylation of target proteins. Since the publication of this work, we have extended the approach to various OST homologs, and have developed high- throughput mass spectrometry testbeds to analyze glycosylation. We anticipate that our technology will enable accelerated enzyme prototyping and expand the available enzyme toolbox for biomanufacturing defined glycoproteins. BIOT 3 Encoding decision-making functions into cell metabolism: the marriage of synthetic biology, metabolic engineering and intelligent control Peng Xu, [email protected]. Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland, United States Living organism is an intelligent system encoded by hierarchically organized information to perform precisely controlled biological functions. With a better understanding of cellular regulation, biomolecular engineers have been able to engineer both the chemistry modules (the mass flow) and the control modules (the information flow) inside the cell to design intelligent cells with desired functions. Instead of programing machine language in a chemical plant, synthetic biologists rewrite the genetic software and encode logic functions in living cells to improve cellular performance. In this lecture, I will present both computational and experimental approach to unravel the design principles underlying efficient biomanufacturing platforms – YIN and Yang DRAFT metabolic balance, autonomous metabolic switches, microbial social interactions for various biotechnological applications. I will present strategies to build genetic toolkits to streamline the genetic/genome modification for a promising industrial yeast Y. lipolytica, which allows us to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA metabolism to produce complex oleochemicals, terpenes, polyketides and aromatic commodity chemicals. By combining metabolic addiction with negative autoregulation, I will also present our recent effort to encode decision-making functions into cell metabolism to partition carbon flux and improve strain stability. Engineering feedback genetic circuits to encode decision-making functions into cell metabolism will present us exciting opportunities to solve the most pressing challenges in health, energy and environment in the 21stcentury. BIOT 4 Quality and quantity?: Enhancing mammalian biomanufacturing performance through systems biotechnology approaches Michael J. Betenbaugh, [email protected]. Department oF Chemical and Biomolecular engineering, Johns Hopkins University, Baltimore, Maryland, United States The traditional engineering paradigm states that you must sacrifice quality for quantity. This can be true for biomanufacturing processes in which quality can involve critical quality attributes such as the glycan structures attached to a produced glycoprotein therapeutic. As biotechnologists, we seek to break this axiom through the application of systems engineering, cell and process modeling, and synthetic biology and metabolic engineering. In our current work we are implementing these systems biotechnology tools to improve the performance of CHO cells and other production hosts. Engineering methodologies are being applied to both determine the glycosylation structures and, as needed, change these glycoforms. One approach being used is to alter the cell lines in order to both add and remove glycosylation capabilities in CHO cells. Determining which modifications are best to implement can be facilitated with the assistance of comprehensive models of glycosylation processes. Another alternative is to adjust the media and examine the role that supplementation with additives has on both the product titer and glycosylation character. It is equally important to be able to evaluate the effect of these engineering modifications and thus analytical tools are needed to determine the glycan patterns for glycoproteins produced in these hosts. Finally, it is also useful to dictate the final glycan structures and this is often best achieved through the use of control strategies based on our DRAFT understanding of cellular glycosylation and the impact of process modifications. Such a comprehensive systems biotechnology approach will enhance our ability to generate desired profiles in terms of glycosylation and other attributes while minimizing the effect on the performance of CHO cells in culture, leading to enhanced production of high quality target biopharmaceuticals in current and future mammalian biomanufacturing processes. BIOT 5 Process development strategy for E. coli based cell-free protein synthesis reactions Noelle Colant1, Jaime Teneb-Lobos1, Stephen Goldrick1, Stefanie Frank1, William Rosenberg2, Daniel G. Bracewell1, [email protected]. (1) Biochemical Engineering, University College London, London, United Kingdom (2) Institute For Liver and Digestive Health, UCL Division of Medicine, London, United Kingdom Over the last decade, cell-free protein synthesis (CFPS) has been utilized as a production platform for antibodies, therapeutic proteins, and vaccine candidates. CFPS is advantageous as a production platform because reactions can achieve relatively high titers in a few hours and reactions have been demonstrated to scale linearly up to 100 L. CFPS reactions also tolerate conditions that are not typically attainable in traditional in vivo cultivations. The open nature of these in vitro systems allows for non-physiological conditions as well as the addition of components not naturally found in E. coli, like non- standard amino acids which can then be incorporated into proteins or chaperones and other agents for post-translational modifications. CFPS process development strategies avoid the need for cell line development but must account for these differences. As CFPS reactions can be completed rapidly we were able to design a rational process development strategy that allows us to optimize titers in ~48 hours. The approach begins by examining the impact of the E. coli strain chosen for the extract, the formulation of the reaction mixture, and the optimization of the plasmid sequence on product titer. Using the system selected
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