Time-Resolved 3D Multi-Resolution Microscopy for Real-Time Cellulase Actions In Situ

Haw Yang, Princeton University (Principal Investigator)

Ming Tien, Penn State University (Co-Investigator)

Cellulose is the most abundant renewable carbon source on earth and thus is of central importance as a biofuels feedstock. A major impediment toward converting it to a liquid fuel is its crystallinity and that it is encased by both lignin and hemicellulose. As such, much research effort has been expended in finding the best cellulases to use for saccharification and how to circumvent the lignin barrier. Little is known with respect to the cellulase mechanism of action, however. This involves initiation (binding of cellulase to cellulase), processive hydrolysis (cyclic rounds of cellobiose release), and termination. Moreover, in this regard, not much is known on how lignin (and hemicellulose) impacts this mechanism. This limitation in knowledge is due, in part, to the substrate being an insoluble polymer for which the tools of conventional biochemistry are not applicable.

The overall goal of this project is to create enabling capabilities for elucidating the kinetic and chemical factors affecting cellulase action on lignocellulose in vitro, and in the future in situ. This will be achieved by the development of time-resolved 3D multi-resolution imaging technology. The 3D capability is essential because in realistic samples, the cell wall / lignocellulose morphology is complex and extends to all three dimensions. The technology continuously tracks the 3D position of a single quantum-dot tagged cellulase with 10-microsecond (μs, 10^-6s) time resolution and ~10-nanometer (nm, 10^-9m) XYZ 3D localization precision. At the same time, the contextual insights for the tracked cellulase are provided by two-photon laser-scanning fluorescence lifetime imaging microscopy.

We will use pure cellulose, lignocellulose (stems of Arabidopsis sectioned) and mutants deficient in either hemicellulose or lignin as substrates for tracking the tagged cellulases. We will obtain kinetic parameters and define the rate-limiting step in cellulase-mediated hydrolysis of cellulose. We will study cellulases from both brown-rot (a moderately processive endoglucanase) and white-rot fungi (exo and endoglucanases). White-rot fungi are able access cellulose and circumvent the lignin barrier through lignin-degrading peroxidases whereas brown-rot fungi are somehow able to circumvent the barrier without degrading it but only modifying it.

With the new time-resolved 3D multi-resolution imaging technology, we will be able to visualize and detect initiation, processive turnover of the enzyme, possible diffusion in a random-walk manner and finally termination. These kinetic steps will be observed with pure cellulose and in the context of lignin and hemicellulose. Our approach will be able to unambiguously follow these mechanism-defining actions of the enzyme due to its nanometer-precision and microsecond 3D tracking. Thus, our research will obtain kinetic parameters and define the rate-limiting step in cellulase-mediated hydrolysis of cellulose.

We plan to achieve the project goal through two aims: (1) To develop a new time-resolved 3D multi-resolution microscope that simultaneously visualizes the dynamics of different scales in situ—the molecular / nanometer scale and the cellular / lignocellulose scale—continuously from μs to minutes and even hours. (2) To critically evaluate the platform by quantitatively resolving the mechanistic steps while the cellulase is in action, one molecule at a time. The technologies will be generalizable to biofuel research areas beyond fungi, extending to the basic science in heterogeneous enzymatic catalysis. Empowered by these new technologies, the community will be able to begin to address those knowledge-gap bridging questions, which in turn can lead to a more predictive understanding of microorganism-enzyme-metabolite synergy.