How Can We Master Energy and Information on the Nanoscale to Create New Technologies with Capabilities Rivaling Those of Living Things? Progress on Grand Challenge New Horizons for Grand Challenge Remaining ChallengeRefreshed Grand Challenge? To harness these discoveries by moving into the realm of synthetic biology and in-vitro reconstitution of synthesizing complex, to engineer new forms of cellulose in a science-based manner. CLSF researchers have made large strides in understanding the machinery underlying cellulose synthesis and also a novel way to connect cellulose microfibrils in plant cell walls. See attached highlights. Has the focus/scope of the Grand Challenge evolved? It is still a good Grand Challenge. Is a new statement of the Grand Challenge needed? Should the Grand Challenge be retired? How about ‘rival or exceed those of living organisms’ Submitted by: Daniel Cosgrove Affiliation: Penn State University

2 In-vitro Reconstitution of Bacterial Cellulose Biosynthesis Significance and Impact This is the first example of cellulose biosynthesis from purified components. It reveals the essential subunits required for function and enables a detailed biochemical analysis of the many reactions required for cellulose synthesis and membrane translocation. Research Details – BcsA is the catalytically active subunit of the cellulose synthase complex but requires BcsB for function. – BcsA synthesizes a cellulose polymer 200 to 300 glucose units in length from UDP-activated glucose. – Cellulose synthesis occurs at a reaction rate of at least 90 glucose units per second. – BcsA is highly specific for UDP-glucose as substrate. – Only the membrane associated region of BcsB is crucial to maintain catalytic activity of BcsA. Omadjela, O., Narahari, Strumillo, J., A., Melida, H., Mazur, O., Bulone, V. & Zimmer, J., BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for cellulose synthesis, PNAS 2013, 110, Scientific Achievement The multi-subunit bacterial cellulose synthase was purified and functionally reconstituted in vitro, producing high molecular weight cellulose.

3 All-atom model of plant cellulose synthase Significance and Impact Our model can be used to explain numerous structure-activity relationships within plant cellulose synthases and may be useful for the selection and subsequent testing of appropriate mutants in order to optimize cellulose and biomass properties. Research Details – Demonstrated that a large (506 amino acid) protein structure can now be now successfully predicted using computational methods – Predicted a conserved mechanism for cellulose catalytic mechanism across Kingdoms – Showed that regions unique to plant CESAs, the CSR and P-CR, fold into distinct subdomains within the cytosolic region, supporting the potential importance of these regions for CESA assembly into plant CSCs. L. Sethaphong, C. H. Haigler, J. D. Kubicki, J. Zimmer, D. Bonetta, S. DeBolt, Y. G. Yingling, "Tertiary model of a plant cellulose synthase", PNAS (2013) 110: Scientific Achievement Predicted a three dimensional structure of the large cytosolic (catalytic) region of a plant cellulose synthase using computational methods

Scientific Achievement Showed that structurally important xyloglucan is not accessible to enzymes that can break it down (xyloglucanases) but instead is intertwined with cellulose in a protected form or compartment Significance and Impact Suggests new ways to loosen cell walls for faster plant growth and for more efficient conversion of plant biomass to biofuels. Also explains why approaches based solely on xyloglucan digestion do not work. Research Details – Measured cell wall biomechanical responses to enzymes that specifically hydrolyze xyloglucan, cellulose, or both. – Xyloglucanases and cellulases, alone or added together, were ineffective for cell wall loosening – Only single enzymes with dual specificities (capable of cutting both xyloglucan and cellulose) were effective in cell wall loosening – Results suggest the common depiction of xyloglucan acting as a tether between cellulose microfibrils is inaccurate A Revised Architecture for Plant Primary Cell Walls Xyloglucans (black lines) were thought to tether cellulose microfibrils (red lines) in 1 o cell walls (top panel). New results indicate a different arrangement, with the load-bearing xyloglucans hidden in tight junctions that bind microfibrils together. Work was performed at Penn State University Y.B. Park, D.J. Cosgrove Plant Physiology 2012, 158, Revised model Tethered network model