Yining Zeng

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.

Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels

A biochemical platform holds the most promising route toward lignocellulosic biofuels, in which polysaccharides are hydrolyzed by cellulase enzymes into simple sugars and fermented to ethanol by microbes. However, these polysaccharides are cross-linked in the plant cell walls with the hydrophobic network of lignin that physically impedes enzymatic deconstruction. A thermochemical pretreatment process is often required to remove or delocalize lignin, which may also generate inhibitors that hamper enzymatic hydrolysis and fermentation. Here we review recent advances in understanding lignin structure in the plant cell walls and the negative roles of lignin in the processes of converting biomass to biofuels. Perspectives and future directions to improve the biomass conversion process are also discussed.

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.

An NAC transcription factor orchestrates multiple features of cell wall development in Medicago truncatula

To identify genes controlling secondary cell wall biosynthesis in the model legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant population for plants with altered patterns of lignin autofluorescence. From more than 9000 R1 plants screened, four independent lines were identified with a total lack of lignin in the interfascicular region. The mutants also showed loss of lignin in phloem fibers, reduced lignin in vascular elements, failure in anther dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls. Microarray and PCR analyses confirmed that the mutations were caused by the insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in different combinations to control cell wall composition in stems and anthers. Loss of MtNST1 function resulted in reduced lignin content, associated with reduced expression of most lignin biosynthetic genes, and a smaller reduction in cell wall polysaccharide content, associated with reduced expression of putative cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase digestion released significantly more sugars from cell walls of nst1 mutants compared with the wild type. We discuss the implications of these findings for the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.

A biophysical perspective on the cellulosome: new opportunities for biomass conversion

The cellulosome is a multiprotein complex, produced primarily by anaerobic microorganisms, which functions to degrade lignocellulosic materials. An important topic of current debate is whether cellulosomal systems display greater ability to deconstruct complex biomass materials (e.g. plant cell walls) than nonaggregated enzymes, and in so doing would be appropriate for improved, commercial bioconversion processes. To sufficiently understand the complex macromolecular processes between plant cell wall polymers, cellulolytic microbes, and their secreted enzymes, a highly concerted research approach is required. Adaptation of existing biophysical techniques and development of new science tools must be applied to this system. This review focuses on strategies likely to permit improved understanding of the bacterial cellulosome using biophysical approaches, with emphasis on advanced imaging and computational techniques.

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.

Elucidating the role of ferrous ion cocatalyst in enhancing dilute acid pretreatment of lignocellulosic biomass

BackgroundRecently developed iron cocatalyst enhancement of dilute acid pretreatment of biomass is a promising approach for enhancing sugar release from recalcitrant lignocellulosic biomass. However, very little is known about the underlying mechanisms of this enhancement. In the current study, our aim was to identify several essential factors that contribute to ferrous ion-enhanced efficiency during dilute acid pretreatment of biomass and to initiate the investigation of the mechanisms that result in this enhancement.ResultsDuring dilute acid and ferrous ion cocatalyst pretreatments, we observed concomitant increases in solubilized sugars in the hydrolysate and reducing sugars in the (insoluble) biomass residues. We also observed enhancements in sugar release during subsequent enzymatic saccharification of iron cocatalyst-pretreated biomass. Fourier transform Raman spectroscopy showed that major peaks representing the C-O-C and C-H bonds in cellulose are significantly attenuated by iron cocatalyst pretreatment. Imaging using Prussian blue staining indicated that Fe2+ ions associate with both cellulose/xylan and lignin in untreated as well as dilute acid/Fe2+ ion-pretreated corn stover samples. Analyses by scanning electron microscopy and transmission electron microscopy revealed structural details of biomass after dilute acid/Fe2+ ion pretreatment, in which delamination and fibrillation of the cell wall were observed.ConclusionsBy using this multimodal approach, we have revealed that (1) acid-ferrous ion-assisted pretreatment increases solubilization and enzymatic digestion of both cellulose and xylan to monomers and (2) this pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by the C-O-C and C-H bonds in cellulose.

Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis

Pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutant of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.