Skip to main content
Photo of Brandon Knott

Brandon Knott

Staff Engineer | 303-384-6223

Research Interests

  • Molecular mechanisms of cellulose biosynthesis

  • Molecular mechanisms of cellulose-degrading enzymes

  • Structure-function relationships in glycoside hydrolases

  • Techno-economic analysis (TEA) of biomass conversion technologies

  • Methane clathrate hydrates formation

Molecular Mechanisms of Cellulose Biosynthesis

Illustration of the domains of the synthase enzyme, represented as  surfaces or embedded in a lipid bilayer represented as yellow multi-jointed strands. Above this is bcsb domain which surrounded by the periplasm. Blob region labeled below are pilz and gt domains. through middle of blobs strand rings. shorter strands attached to

Cellulose is the primary component of plant cell walls and the world's most abundant biopolymer, with several billion tons biosynthesized annually. The molecular mechanism by which this is accomplished by cellulose synthase enzymes is a mystery that has only begun to be revealed. The first crystal structure of a functional cellulose synthase was published in 2013, in this case from a bacterial system. The bacterial cellulose synthase (Bcs, see figure) is a membrane-embedded enzyme complex that comprises (1) a glycosyltransferase (GT) domain where the chemical reaction occurs, (2) transmembrane (TM) helices through which the nascent chain is translocated across the lipid bilayer, (3) a PilZ domain that binds cyclic-di-GMP (which allosterically regulates bacterial cellulose biosynthesis), and (4) a periplasmic B domain (BcsB). The publication of this structure has opened up the possibility of deploying molecular simulation to study the molecular details of cellulose biosynthesis.

Molecular Mechanisms of Cellulose-Degrading Enzymes

Illustration of the domains of the cellobiohydrolase enzyme, represented as  surfaces or on the surface of cellulose represented as three layers green and red multi-jointed strands one these is pulled out threaded through middle domain labeled domain. this attached to a much smaller more cylindrical which in turn linked roughly spherical module

The degradation of cellulose by glycoside hydrolase enzymes has been studied more extensively than its biosynthesis, but important questions still remain. Cellobiohydrolases are cellulase enzymes that processively hydrolyze individual cellulose chains, producing cellobiose (a dimer of glucose). Cellobiohydrolases complete a "processive cycle" repeatedly before disengaging from the chain; this cycle includes several steps, which are extremely difficult to differentiate experimentally. We employ advanced molecular simulation tools to study each step individually (including the chemical reaction and cellulose processivity). For example, we study Trichoderma reesei Cel7A (see figure), the most prevalent component of industrial cellulase mixtures and an effective model system for studying processive cellulase enzymes. Taken together with past studies, our results shed light on rate-limiting steps in cellobiohydrolases, providing potential protein engineering targets.

Structure-Function Relationships in Glycoside Hydrolases

Illustration of several different enzymes, which appear as  blobs. each enzyme has a multi-jointed strand of blue and red rings representing cellulose oligomers. one blob nearly completely encloses this whereas another leaves it exposed visible the top shown at bottom are four these enzymes that expose increasing more in order: trcel7a pccel7d trcel5a trcel12a

As detailed in our group's recent Chemical Reviews article, we are keenly interested in developing and understanding structure-function relationships in enzymes important to the breakdown of biomass. These relationships can help to guide protein engineering efforts. Comparing cellobiohydrolases (CBHs) and endoglucanases (EGs) provides one example of this type of relationship (see figure). The primary structural difference between TrCel7A (a CBH, left-most figure in all three rows, shown in green) and TrCel7B (an EG, right-most panel in the top two rows, shown in light orange) are the loops that cover the enzymes active site. This structural difference has been shown to have a dramatic consequence for the function of these enzymes, including their processive ability (i.e., the number of bonds they hydrolyze before dissociating from a cellulose chain) and their probability of endo-initiation (i.e., hydrolyzing a bond in the interior of the cellulose chain, rather than at the chain end). Also shown are P. chrysosporium Cel7D (in blue, a CBH with less extensive loops than TrCel7A) and endoglucanases TrCel5A and TrCel12A.

Techno-Economic Analysis of Biomass Conversion Technologies

Techno-economic analysis (TEA) is capable of examining both the current and the potential viability of a given pre-commercial technology. At the National Renewable Energy Laboratory (NREL), TEA also helps to direct research effort into those areas where there is the greatest potential for increasing the cost-competitiveness of a given technology (see NREL's design reports on possible thermochemical and biochemical conversion routes to hydrocarbons from lignocellulosic biomass). I am currently involved in TEA projects applied to both biochemical and thermochemical platforms for transforming biomass into fuels and value-added co-products.

Methane Clathrate Hydrates Formation

Graph of  free energy as a function of nucleus size showing parabolic curve with maximum labeled embedded on this graph is an illustration roughly spherical agglomeration and vertex cages. Each these cages contains black sphere. there are additional spheres at the surface that not in four squiggly arrows shown pointing toward two away.

Clathrate hydrates are multi-component crystalline inclusion compounds of hydrogen-bonded water "cages" surrounding a hydrophobic guest molecule. Methane hydrates are a particularly important example, as they occur in vast natural reservoirs around the globe and have relevance to global climate trends, carbon dioxide sequestration, and gas storage and transportation. Perhaps most significantly, they can threaten oil and natural gas production by blocking pipelines and exacerbating oil-well blowouts. With collaborators at the Center for Hydrate Research at the Colorado School of Mines, we have utilized molecular simulation to investigate the molecular mechanism by which these compounds form. The figure shows a schematic representation of the nucleation of a hydrates nucleus. Red, green, and blue lines represent hydrogen bonds between water molecules in different types of cages surrounding methane molecules (black spheres).


  • Ph.D., Chemical Engineering, University of California, Santa Barbara, 2012

  • B.S., Chemical Engineering, Arizona State University, 2007

Professional Experience

  • Postdoctoral Researcher, NREL, National Bioenergy Center (NBC), 2014–present

  • Director's Postdoctoral Fellow, NREL, NBC, 20122014

  • Graduate Intern, The Boeing Company, 2011

  • Undergraduate Intern, Freescale Semiconductor, 2006

Featured Publications

  1. "Simulations of cellulose translocation in the bacterial cellulose synthase suggest a regulatory mechanism for the dimeric structure of cellulose," Chemical Science (2016)

  2. "Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases," Chemical Science (2016)

  3. "Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life," Applied and Environmental Microbiology (2016)

  4. "Fungal Cellulases," Chemical Reviews (2015)

  5. "Nucleation rate analysis of methane hydrate from molecular dynamics simulations," Faraday Discussions (2015)

  6. "Reaction coordinate of incipient methane clathrate hydrate nucleation," The Journal of Physical Chemistry B (2014)

  7. "Carbohydrate-protein interactions that drive processive polysaccharide translocation in enzymes revealed from a computational study of cellobiohydrolase processivity," Journal of the American Chemical Society (2014)

  8. "Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases," Current Opinion in Biotechnology (2014)

  9. "The mechanism of cellulose hydrolysis by a two-step, retaining cellobiohydrolase elucidated by structural and transition path sampling studies," Journal of the American Chemical Society (2013)

  10. "Homogeneous nucleation of methane hydrates: unrealistic under realistic conditions," Journal of the American Chemical Society (2012)

  11. "Bubbles, crystals, and laser - induced nucleation," Journal of Chemical Physics (2011)

  12. "A simulation test of the optical Kerr mechanism for laser-induced nucleation," Journal of Chemical Physics (2011)

  13. "Estimating diffusivity along a reaction coordinate in the high friction limit: Insights on pulse times in laser-induced nucleation," Journal of Chemical Physics (2009)

View all NREL Publications for Brandon Knott.