Available online at www.sciencedirect.com A biophysical perspective on the cellulosome: new opportunities for biomass conversion Shi-You Ding 1 , Qi Xu 1 , Michael Crowley 1 , Yining Zeng 1 , Mark Nimlos 2 , Raphael Lamed 3 , Edward A Bayer 4 and Michael E Himmel 1 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. Address 1 Chemical and Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401, USA 2 National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, USA 3 Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel 4 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author: Ding, Shi-You (shi_you_ding@nrel.gov) Current Opinion in Biotechnology 2008, 19:218–227 This review comes from a themed issue on Energy Biotechnology Edited by Lee R. Lynd Available online 29th May 2008 0958-1669/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.copbio.2008.04.008 Introduction Biological conversion of lignocellulosic materials has been proposed as a sustainable and renewable route for the production of liquid transportation fuels [1  ]. Current technology for biomass conversion to biofuels, primarily bioethanol, involves the integration of three major unit operations (steps): particle size reduction and pretreat- ment, enzymatic hydrolysis, and fermentation of the lignocellulosic sugars. Pretreatment of biomass feed- stocks produces materials that are more amenable to enzymatic digestion, often involving chemical treatment at a temperature range of 120–200 8C. Inoculation of the biomass during storage with microbial communities has also been proposed as a means of reducing required pretreatment severity [2]. In such processes, thermal/ chemical pretreatment hydrolyzes easily available hemi- celluloses, rendering the feedstock accessible to cellu- lases and hemicellulases, which catalyze enzymatic hydrolysis to soluble sugars. These sugars are subjected to fermentation for bioethanol production in a myriad of varied processing schemes. Enzymatic hydrolysis is often considered the feasibility-limiting step, because of the high cost and limited performance of currently available enzyme preparations. Indeed, current processing strat- egies have been derived empirically, with little knowl- edge of the fine structure of the feedstocks and even less information about the molecular processes involved in biomass conversion. Substantial progress toward cost- effective conversion of biomass to fuels would be fostered by fundamental breakthroughs in our current understand- ing of the chemical and structural properties that have evolved in the plant cell walls, which prevent its easy disassembly, collectively known as ‘biomass recalci- trance.’ Recently, new strategies in biotechnology have been pursued to reduce the cost of the cellulases used for biomass conversion. Most actual improvements in proces- sing cost have come from work to improve enzyme productivity, not enzyme performance. Improvements in cellulase performance have been incremental, when reported, include engineering enzyme component mix- tures (i.e. for superior synergism), enzyme robustness (usually assured when enzymes from thermophiles are used), and processing options designed to be synergistic, that is, simultaneous saccharification and fermentation (SSF). In nature, there are currently two major types of cellu- lolytic systems recognized, those based on ‘free’ enzymes that are discretely acting cellulases typically produced by aerobic fungi and bacteria and those based on complexes of cellulolytic enzymes or ‘cellulosomes’ produced by some anaerobic bacteria. An important concept currently debated is whether or not cellulosomal systems display greater ability to deconstruct complex biomass materials, such as the plant cell walls, than do noncomplexed enzymes. For example, some evidence suggests that Current Opinion in Biotechnology 2008, 19:218–227 www.sciencedirect.com