Yu San Liu

How Does Plant Cell Wall Nanoscale Architecture Correlate with Enzymatic Digestibility

One of the key challenges in scaling up biofuels manufacturing is development of a cost-effective way to break down cellulose into sugars for subsequent fermentation. Ding et al. (p. 1055) applied several different types of microscopy to understand the details of how cellulase enzymes perform this task, in the interest of ultimately optimizing the procedure. After lignin removal, fungal cellulases penetrated the remaining cellulose pore structure more efficiently than did bacteria-derived multienzyme complexes. However, this behavior hinges on a lignin extraction scheme that preserves the native architecture of the cellulose. Microscopy techniques uncover the distinct mechanisms of different enzyme classes in breaking down cellulose for biofuels. Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels. Here, we report the use of correlative imaging in real time to assess the impact of pretreatment, as well as the resulting nanometer-scale changes in cell wall structure, upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes (cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially native microfibrillar structure.

Cellobiohydrolase Hydrolyzes Crystalline Cellulose on Hydrophobic Faces

Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.

Label-Free, Real-Time Monitoring of Biomass Processing with Stimulated Raman Scattering Microscopy

Research into alternative energy has experienced dramatic growth in recent years, which was motivated by both the environmental impact of current fossil fuels and the unstable and uncertain sources of oil and natural gas. Under ideal conditions, currently unused plant materials, such as agricultural residues, forestry wastes, and energy crops, can be broken down by a series of chemical, enzymatic, and/or microbiological processes into ethanol or other biofuel sources. Biofuels offer an infinitely renewable source of carbon-neutral fuels that can be produced domestically and can make use of waste products from agricultural activity already taking place. The major challenge to be overcome in the widespread adoption of many biofuels is that biomass is intrinsically recalcitrant, making conversion into usable fuels inefficient. This, in turn, means that substantial energy is required to produce the current generation of biofuels, thus decreasing or eliminating their advantages as alternative sources of fuel. The two major chemical species of interest in the biomass conversion process are lignins and polysaccharides such as cellulose and hemicelluloses. Lignins are partly responsible for biomass recalcitrance, but they may also have value as side products in the biorefineries of the future. Cellulose can be broken down to simple sugars, which can then be fermented to produce ethanol. To address the recalcitrance problem presented by lignins, a thermochemical pretreatment process is necessary in current biomass conversion technology. This process uses oxidizing, acidic, or basic conditions along with elevated pressures and/or temperatures to remove or modify lignins and hemicelluloses, thereby enhancing the accessibility for the cellulase enzymes used in the breakdown of cellulose. 6] To optimize the overall conversion efficiency, a detailed understanding of the hydrolysis kinetics of polysaccharides and lignins is critical. For this reason, analytical tools to study the biomass conversion process are needed. Herein, we demonstrate that stimulated Raman scattering (SRS) microscopy, a new imaging method, can offer new information on the biomass conversion processes. The ideal technique for studying the conversion process in situ should offer chemical specificity without exogenous labels, non-invasiveness, high spatial resolution, and real-time monitoring capability. Current analytical methods, such as gas chromatography–mass spectrometry, electron or scanning-probe microscopy, and fluorescence microscopy, cannot satisfy all of these requirements. Microscopy based on infrared absorption offers chemical specificity, but the spatial resolution is limited by the long infrared wavelengths, and penetration depth into aqueous plant samples is limited. Raman microspectroscopy is widely used because it offers label-free chemical contrast with high resolution and chemical specificity. However, the Raman scattering effect is weak, and long pixel dwell times (on the order of 0.1–1 s) are required for imaging plant materials. This means that real-time imaging is challenging, as even a 256! 256 pixel image would require almost two hours at 0.1 s/pixel. Consequently, the dynamic processes involved in the conversion cannot be followed at high spatiotemporal resolution. Coherent Raman microscopy techniques solve many of these problems and offer label-free chemical imaging with high sensitivity and high spatial resolution. Coherent antiStokes Raman scattering (CARS) microscopy is a technique that has been developed over the past ten years and applied to numerous problems of biological or biomedical relevance. However, CARS microscopy suffers from a nonresonant electronic background that can distort the chemical information of interest, making quantitative image interpretation challenging. Herein, we introduce stimulated Raman scattering (SRS) as a tool to study biomass conversion. SRS [*] B. G. Saar, Prof. X. S. Xie Department of Chemistry and Chemical Biology Harvard University, Cambridge, MA (USA) Fax: (+1)617-496-8709 E-mail: xie@chemistry.harvard.edu Y. Zeng, Y. Liu, M. E. Himmel, S. Ding Biosciences Center, National Renewable Energy Laboratory Golden, CO (USA) and Bioenergy Science Center, Oak Ridge National Laboratory Oak Ridge, TN (USA) Fax: (+1)303-384-7752 E-mail: shi.you.ding@nrel.gov C. W. Freudiger Department of Physics and Department of Chemistry and Chemical Biology Harvard University, Cambridge, MA (USA) [**] We thank G. R. Holtom and M. B. J. Roeffaers for helpful discussions. B.G.S. was supported by the Army Research Office through an NDSEG fellowship. C.W.F. was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship. This work is also supported by the US Department of Energy: the instrumentation and data analysis is funded under grant DE-FG02-07ER64500, and the BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological & Environmental Research in the DOE Office of Science; the delignification process is funded by the Office of the Biomass Program. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201000900. Communications

In situ imaging of single carbohydrate-binding modules on cellulose microfibrils

The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates, facilitated by surface absorption of the carbohydrate-binding modules (CBMs), a component of most cellulase systems. Despite their importance, reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here, we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment, using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril, with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes, thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.

Imaging lignin-downregulated alfalfa using coherent anti-Stokes Raman scattering microscopy

Targeted lignin modification in bioenergy crops could potentially improve conversion efficiency of lignocellulosic biomass to biofuels. To better assess the impact of lignin modification on overall cell wall structure, wild-type and lignin-downregulated alfalfa lines were imaged using coherent anti-Stokes Raman scattering (CARS) microscopy. The 1,600-cm−1 Raman mode was used in CARS imaging to specifically represent the lignin signal in the plant cell walls. The intensities of the CARS signal follow the general trend of lignin contents in cell walls from both wild-type and lignin-downregulated plants. In the downregulated lines, the overall reduction of lignin content agreed with the previously reported chemical composition. However, greater reduction of lignin content in cell corners was observed by CARS imaging, which could account for the enhanced susceptibility to chemical and enzymatic hydrolysis observed previously.

A single molecule study of cellulase hydrolysis of crystalline cellulose

Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

Imaging Cellulose Using Atomic Force Microscopy

Cellulose is an important biopolymer primarily stored as plant cell wall material. Plant-synthesized cellulose forms elementary fibrils that are micrometers in length and 3-5 nm in dimensions. Cellulose is a dynamic structure, and its size and property vary in different cellulose-containing materials. Atomic force microscopy offers the capability of imaging surface structure at the subnanometer resolution and under nearly physiological conditions, therefore providing an ideal tool for cellulose characterization.

DOPI and PALM imaging of single carbohydrate binding modules bound to cellulose nanocrystals

We use single molecule imaging methods to study the binding characteristics of carbohydrate-binding modules (CBMs) to cellulose crystals. The CBMs are carbohydrate specific binding proteins, and a functional component of most cellulase enzymes, which in turn hydrolyze cellulose, releasing simple sugars suitable for fermentation to biofuels. The CBM plays the important role of locating the crystalline face of cellulose, a critical step in cellulase action. A biophysical understanding of the CBM action aids in developing a mechanistic picture of the cellulase enzyme, important for selection and potential modification. Towards this end, we have genetically modified cellulose-binding CBM derived from bacterial source with green fluorescent protein (GFP), and photo-activated fluorescence protein PAmCherry tags, respectively. Using the single molecule method known as Defocused Orientation and Position Imaging (DOPI), we observe a preferred orientation of the CBM-GFP complex relative to the Valonia cellulose nanocrystals. Subsequent analysis showed the CBMs bind to the opposite hydrophobic <110> faces of the cellulose nanocrystals with a welldefined cross-orientation of about ~ 70°. Photo Activated Localization Microscopy (PALM) is used to localize CBMPAmCherry with a localization accuracy of ~ 10nm. Analysis of the nearest neighbor distributions along and perpendicular to the cellulose nanocrystal axes are consistent with single-file CBM binding along the fiber axis, and microfibril bundles consisting of close packed ~ 20nm or smaller cellulose microfibrils.

Single-Molecule Tracking of Carbohydrate-Binding Modules on Cellulose Using Fluorescence Microscopy

Single-molecule fluorescence detection is an invaluable technique for the study of molecular behavior in biological systems, both in vitro and in vivo. In this chapter, we focus on detailed protocols that utilize Total Internal Reflection Fluorescence Microscopy (TIRF-M) to visualize single molecules of carbohydrate-binding module (CBM) labeled with green fluorescent protein (GFP). The content describes step-by-step sample preparation and data acquisition, processing, and analysis. These methods can also be further used to study interactions between domains of cellulase molecules and between cellulases and cellulose.