1 Introduction……………………………………………………………………………………………… 206

2 Baker’s Yeast (S. cerevisiae) as a CBP Host…………………………………………………….. 208

3 Engineering S. cerevisiae for Sugar Fermentation……………………………………………… 210

4 Expression of Cellulases in S. cerevisiae…………………………………………………………. 211

5 Expression of Hemicellulases in S. cerevisiae…………………………………………………… 218

6 Selection for the Development of Superior CBP Yeasts………………………………….. 224

7 Integration of Different Enzymatic Activities

into a Single CBP Yeast and Transfer to Industrial Strains………………………. 228

References…………………………………………………………………………………………………….. 230

Abstract Consolidated bioprocessing (CBP) of lignocellulose to bioethanol refers to the combining of the four biological events required for this conversion process (production of saccharolytic enzymes, hydrolysis of the polysaccharides present in pretreated biomass, fermentation of hexose sugars, and fermentation of pentose sugars) in one reactor. CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass pro­cessing. Although no natural microorganism exhibits all the features desired for CBP, a number of microorganisms, both bacteria and fungi, possess some of the desirable properties. This review focuses on progress made toward the development of baker’s yeast (Saccharomyces cerevisiae) for CBP. The current status of saccharolytic enzyme (cellulases and hemicellulases) expression in S. cerevisiae to complement its natural fermentative ability is highlighted. Attention is also devoted to the challenges ahead to integrate all required enzymatic activities in an industrial S. cerevisiae strain(s) and the need for molecular and selection strategies pursuant to developing a yeast capable of CBP.

Keywords Consolidated bioprocessing • Cellulolytic yeast •

One-step bioethanol production • Saccharomyces cerevisiae

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Introduction

Biomass is the only foreseeable renewable feedstock for sustainable produc­tion of biofuels. The main technological impediment to more widespread utilization of this resource is the lack of low-cost technologies to overcome the recalcitrance of the cellulosic structure [1]. Four biological events occur during conversion of lignocellulose to ethanol via processes featuring enzy­matic hydrolysis: production of saccharolytic enzyme (cellulases and hemi — cellulases), hydrolysis of the polysaccharides present in pretreated biomass, fermentation of hexose sugars, and fermentation of pentose sugars [2]. The hydrolysis and fermentation steps have been combined in simultaneous sac­charification and fermentation (SSF) of hexoses and simultaneous saccharifi­cation and cofermentation (SSCF) of both hexoses and pentoses schemes. The ultimate objective would be a one-step “consolidated” bioprocessing (CBP) of lignocellulose to bioethanol, where all four of these steps occur in one re­actor and are mediated by a single microorganism or microbial consortium able to ferment pretreated biomass without added saccharolytic enzymes (Fig. 1).

CBP is gaining increasing recognition as a potential breakthrough for low — cost biomass processing. A fourfold reduction in the cost of biological pro­cessing and a twofold reduction in the cost of processing overall is projected when a mature CBP process is substituted for an advanced SSCF process fea­turing cellulase costing US $0.10 per gallon ethanol [3]. The US Department of Energy (DOE) Biomass Program multiyear technical plan states: “Mak­ing the leap from technology that can compete in niche or marginal markets for fuels and products also requires expanding the array of possible concepts and strategies for processing biomass. Concepts such as consolidated bio­processing… offer new possibilities for leapfrog improvements in yield and cost.” [4]. The detailed analysis of mature biomass conversion processes by Greene et al. [5] finds CBP to be responsible for the largest cost reduction of all R&D-driven improvements incorporated into mature technology scenar­ios featuring projected ethanol selling prices of less than US $0.70 per gallon. Finally, a recent report entitled Breaking the Biological Barriers to Cellulosic Ethanol states: “CBP is widely considered to be the ultimate low-cost configu­ration for cellulose hydrolysis and fermentation.” [6].

In addition to being desirable, recent studies of naturally occurring cel­lulolytic microorganisms provide increasing indications that CBP is feas­ible. Lu et al. [7] showed that cellulase-specific cellulose hydrolysis rates exhibited by growing cultures of Clostridium thermocellum exceed specific rates exhibited by the Trichoderma reesei cellulase system by approximately 20-fold, with a substantial part of this difference resulting from “enzyme — microbe synergy” involving enhanced effectiveness of cellulases acting as part of cellulose-enzyme-microbe complexes. Whereas cellulase synthesis

was thought to be a substantial metabolic burden for anaerobic microbes fermenting cellulose without added saccharolytic enzymes, C. thermocellum realizes cellulose-specific bioenergetic benefits that exceed the bioenergetic cost of cellulase synthesis [8]. These and other observations provide guid­ance with respect to features that may be beneficial in the course of creating recombinant cellulolytic microbes, and also underscore the point that micro­bial cellulose utilization is differentiable from enzymatic hydrolysis from both fundamental and applied perspectives [1,3].

Although no natural microorganism exhibits all the features desired for CBP, a number of microorganisms, both bacteria and fungi, possess some of the desirable properties. These microorganisms can broadly be divided into two groups: (1) native cellulolytic microorganisms that possess supe­rior saccharolytic capabilities, but not necessarily product formation, and

(2) recombinant cellulolytic microorganisms that naturally give high prod­uct yields, but into which saccharolytic systems need to be engineered [1, 9]. Examples of native cellulolytic microorganisms under consideration in­clude anaerobic bacteria with highly efficient complexed saccharolytic sys­tems, such as mesophilic and thermophilic Clostridium species [9,10], and fungi that naturally produce a large repertoire of saccharolytic enzymes, such as Fusarium oxysporum [11] and a Trichoderma species [12]. However, the anaerobic bacteria produce a variety of fermentation products, limiting the ethanol yield, whereas the filamentous fungi are slow cellulose degraders and give low yields of ethanol [13]. Candidates considered as potential re­combinant cellulolytic microorganisms into which saccharolytic systems have been engineered include the bacteria Zymomonas mobilis [14,15], Escherichia coli [16,17] and Klebsiella oxytoca [18,19], and the yeast Saccharomyces cere — visiae and xylose-fermenting yeasts Pachysolen tannophilus [20], Pichia stipi — tis, and Candida shehatae [21].

While both native and recombinant cellulolytic microorganisms merit in­vestigation, this review will focus on the well-known ethanol producing yeast S. cerevisiae, which has a long commercial history as microorganism of choice for beer, wine, baker’s yeast, and commercial ethanol production. In particu­lar, we address recent progress in heterologous cellulase expression pursuant to development of recombinant cellulose-fermenting yeast strains [22-25].

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