Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing

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Biotechnology Advances xxx (2013) xxx–xxx

JBA-06655; No of Pages 10

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Biotechnology Advances

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Research review paper

Endowing non-cellulolytic microorganisms with cellulolytic activity aiming forconsolidated bioprocessing

Ryosuke Yamada a, Tomohisa Hasunuma a, Akihiko Kondo b,⁎a Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japanb Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

⁎ Corresponding author. Tel./fax: +81 78 803 6196.E-mail address: [email protected] (A. Kondo).

0734-9750/$ – see front matter © 2013 Published by Elhttp://dx.doi.org/10.1016/j.biotechadv.2013.02.007

Please cite this article as: Yamada R, et abioprocessing, Biotechnol Adv (2013), http:/

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Keywords:Consolidated bioprocessCellulolytic microorganismsCellulaseLignocellulosic biomassBiofuelsBio-based chemicalsMolecular engineeringCell surface engineering

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RWith the exhaustion of fossil fuels and with the environmental issues they pose, utilization of abundantlignocellulosic biomass as a feedstock for biofuels and bio-based chemicals has recently become an attractiveoption. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin and has a veryrigid and complex structure. It is accordingly much more expensive to process than starchy grains becauseof the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis.Efficient and cost-effective methods for the production of biofuels and chemicals from lignocellulose arerequired. A consolidated bioprocess (CBP), which integrates all biological steps consisting of enzyme produc-tion, saccharification, and fermentation, is considered a promising strategy for reducing production costs.Establishing an efficient CBP using lignocellulosic biomass requires both lignocellulose degradation intoglucose and efficient production of biofuels or chemicals from glucose. With this aim, many researchers areattempting to endow selected microorganisms with lignocellulose-assimilating ability. In this review, wefocus on studies aimed at conferring lignocellulose-assimilating ability not only to yeast strains but also tobacterial strains by recombinant technology. Recent developments in improvement of enzyme productivityby microorganisms and in improvement of the specific activity of cellulase are emphasized.

© 2013 Published by Elsevier Inc.

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Contents

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RR1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2. Recombinant cellulolytic microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1. Degradation of cellulose by three types of cellulases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Cellulase expression in bacterial strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3. Cellulase expression in yeast strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Cellulase overexpression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1. Secretion versus cell surface display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Overexpression strategies of cellulase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Molecular engineering of cellulases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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1. Introduction

With the exhaustion of fossil fuels and with the environmental is-sues they pose, including global warming and acid rain, utilization ofbiomass as a feedstock for the production of biofuels and bio-based

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l, Endowing non-cellulolytic/dx.doi.org/10.1016/j.biotech

chemicals has recently become an attractive option. Currently, themain feedstock for biofuel production is starch-rich biomass, whichis rapidly hydrolyzed by amylases to give high yields of glucose. How-ever, lignocellulosic biomass such as sugar cane bagasse, corn stover,rice and wheat straw, switchgrass, and poplar is considered a promis-ing starting material for biofuels and chemicals production, owing toits abundance, low expense, renewability, and favorable environmen-tal properties. Lignocellulose is primarily composed of cellulose,

microorganisms with cellulolytic activity aiming for consolidatedadv.2013.02.007

http://dx.doi.org/10.1016/j.biotechadv.2013.02.007

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hemicellulose, and lignin. Cellulose, the main framework of lignocel-lulose, is composed of chains of glucose molecules linked by β-1,4bonds. These chains are in turn linked by hydrogen bonds, makingthe structure very rigid. The cellulose chains are enclosed by hemicel-lulose and lignin. The structure of hemicellulose is more complexthan that of cellulose and contains xylose, mannose, galactose, arabi-nose, acetate, glucuronic acid, and other components. The structure oflignin is also complex. It is composed of phenylpropane units such asp-coumaryl, coniferyl, guaiacyl, syringyl, and sinapyl alcohols (VanDyk and Pletschke, 2012). As described above, lignocellulose has avery rigid and complex structure. For this reason, its conversion ismuch more expensive than that of starchy grains, because of theneed for extensive pretreatment and relatively large amounts of cel-lulases for effective hydrolysis. Thus, efficient and cost-effectivemethods for the degradation of lignocellulose are required for ligno-cellulosic biorefineries.

The production of biofuels and bio-based chemicals by microorgan-isms consists of the following steps: (1) chemical and/or physicochem-ical pretreatment to swell the biomass, (2) enzyme production bymicroorganisms such as filamentous fungi for hydrolyzing the biomass,(3) enzymatic hydrolysis of pretreated biomass to fermentable sugars,(4) fermentation of hexoses derivedmainly from cellulose and pentosesderived mainly from hemicellulose (Hasunuma and Kondo, 2012). Asshown in Fig. 1, the most classic process is called separate hydrolysisand fermentation (SHF). In this process, each step conducted separatelymay be optimized individually. However, the multiple reaction steps inSHF incur high facility and operating costs. To simplify and reduce pro-duction costs, simultaneous saccharification and fermentation (SSF)was developed. In this process, saccharification and fermentation areconducted in one reactor, reducing the cost of facilities and operation.

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Biomass

Pretreatment

Enzyme production

Enzyme production

Saccharification

Saccharification

Biomass

Pretreatment

Enzyme prod

SaccharificHexose and Pentose

Biomass

Pretreatment Enzyme production, SHexose and Pentos

Simultaneous saccharif

Separate hydrolysis an

Consolidated bioproces

Fig. 1. Biofuel and bio-based che

Please cite this article as: Yamada R, et al, Endowing non-cellulolyticbioprocessing, Biotechnol Adv (2013), http://dx.doi.org/10.1016/j.biotech

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However, optimum temperature and pH for saccharification and fer-mentation are different, and make control of SSF difficult. The most in-tegrated process is called consolidated bioprocess (CBP). In CBP, allbiological processes including enzyme production, saccharification,and hexose and pentose fermentation are conducted in a single reactorby a single microorganism. Although optimization of CBP and develop-ment of ideal microorganisms are difficult, the cost of facilities and op-eration is expected to be drastically reduced. Recent research hasprovide considerable attention on process optimization and/or devel-opment of microorganisms for CBPs designed for efficient productionof biofuels and chemicals (la Grange et al., 2010; Lynd et al., 2005;Tamaru et al., 2010; Xu et al., 2009).

The design of an efficient CBP for lignocellulosic biomass requires boththe degradation of lignocellulose into monosaccharides such as glucoseand xylose, and the production of large amounts of biofuels or chemicalsfrom these monosaccharides. Species known to assimilate lignocellulosicbiomass naturally include the filamentous fungus Trichoderma reesei andanaerobic clostridial bacteria. Approaches that have succeeded in confer-ring on native cellulolytic microorganisms the ability to produce high-value products have been summarized in previous reviews (Maki et al.,2009; Peterson and Nevalainen, 2012; Tamaru et al., 2010). However,many researchers are attempting to confer lignocellulose-assimilatingability on various industrial non-cellulolytic microorganisms.

It has already been reviewed to develop recombinant cellulolyticyeast S. cerevisiae for ethanol production from lignocellulose in detailelsewhere (Hasunuma and Kondo, 2012; Laluce et al., 2012). In this re-view, we focus on research aimed at conferring cellulose-assimilatingability by recombinant technology on non-cellulolytic strains of yeastand bacteria. Furthermore, recent developments in the improvementof cellulase productivity by microorganisms and in the improvement

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Biofuelsand chemicals

Hexosefermentation

Pentosefermentation


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ation, fermentation


accharification,e fermentation

ication and fermentation process

d fermentation process

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mical production schemes.



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of the specific activity of cellulase bymolecular engineering strategy arealso emphasized.

2. Recombinant cellulolytic microorganisms

2.1. Degradation of cellulose by three types of cellulases

In general, three types of cellulases are required for the productionof glucose monomers from cellulose, the major component of ligno-cellulosic biomass (Van Dyk and Pletschke, 2012). Endoglucanase(EC 3.2.1.4) degrades the non-crystalline region of the cellulosechain. Subsequently, enzyme called cellobiohydrolases (EC 3.2.1.91and EC 3.2.1.176) attack the exposed ends of the cellulose chainsand degrade its crystalline region. Through the synergistic action ofthese enzymes, cellulose is degraded into cellooligosaccharide. Final-ly, β-glucosidases (EC 3.2.1.21) degrade cellooligosaccharide into glu-cose. Cellulose is degraded by the synergistic action of these threetypes of enzymes. As summarized in Table 1, to degrade celluloseinto glucose and to produce high-value compounds, these enzyme

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Table 1Recombinant cellulolytic microorganisms.

Host strain Cellulosic materials for growthand/or fermentation

Expressing ce

Bacterial strainKlebsiella oxytoca Crystalline cellulose Endoglucana

with low levK. oxytoca Amorphous cellulose EndoglucanaZymomonas mobilis Cellobiose β-glucosidasZymobacter palmae Cellobiose β-glucosidasLactobacillus plantarum β-glucan Endoglucana

with β-glucoZ. mobilis CMC and pretreated bagasse EndoglucanaBacillus subtilis Regenerated amorphous cellulose

and retreated lignocellulosesEndoglucana

Corynebacterium glutamicum β-glucan Endoglucanaβ-glucosidas

Escherichia coli Cellobiose β-glucosidasE. coli Phosphoric acid-swollen cellulose

and pretreated corn stoverEndoglucanaβ-glucosidas

Yeast strainSaccharomyces cerevisiae Cellobiose β-Glucosidas

from ButyriviPhanerochaetfrom R. flavef

S. cerevisiae β-glucan EndoglucanaA. aculeatus

S. cerevisiae Phosphoric acid-swollen cellulose Endoglucanaand β-glucos

S. cerevisiae Phosphoric acid-swollen cellulose EndoglucanaSaccharomyc

Kluyveromyces marxianus CMC and cellobiose Endoglucanaβ-glucosidas

S. cerevisiae β-glucan and cellobiose EndoglucanaS. cerevisiae β-glucan Endoglucana

A. aculeatusS. cerevisiae Phosphoric acid-swollen cellulose Endoglucana

C. cellulolyticS. cerevisiae Phosphoric acid-swollen cellulose Endoglucana

β-glucosidasS. cerevisiae Phosphoric acid-swollen cellulose Endoglucana

β-glucosidasK. marxianus β-glucan Endoglucana

A. aculeatusS. cerevisiae Phosphoric acid-swollen cellulose Endoglucana

β-glucosidasS. cerevisiae β-glucan, acid-treated Avicel,

and cassava pulpα-Amylase frRhizopus oryzT. reesei, and

S. cerevisiae Phosphoric acid-swollen celluloseand pretreated rice straw

Endoglucanaβ-glucosidas

S. cerevisiae Phosphoric acid-swollen cellulose Endoglucanafrom C. therm


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genes may be expressed, via recombination technology, in varioustypes of microorganisms.

2.2. Cellulase expression in bacterial strains

Zhou et al. (2001), Zhou and Ingram (2001) have reported the ex-pression of endoglucanase from Erwinia chrysanthemi in Klebsiellaoxytoca. Because the K. oxytoca strain used in that study could naturallyassimilate cellooligosaccharide, the resulting strain could directly as-similate amorphous cellulose. These researchers further integratedgenes for alcohol dehydrogenase and pyruvate decarboxylase fromthe ethanol-producing bacterium Zymomonas mobilis into the genomeof the recombinant cellulolytic K. oxytoca, and finally achieved directethanol fermentation from amorphous cellulose at 58%–76% of the the-oretical yield.

Some research groups have succeeded in conferring cellulolyticability on ethanol-producing bacterial species of the genus Zymobacterand Zymomonas. Yanase et al. (2005a, b) successfully expressed theβ-glucosidase gene from the cellulolytic rumen bacterium Ruminococcus

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llulase Reference

se from Erwinia chrysanthemiels of commercial cellulase

Zhou et al. (2001a)

se from E. chrysanthemi Zhou and Ingram (2001b)e from Ruminococcus albus Yanase et al. (2005a)e from R. albus Yanase et al. (2005b)se from Clostridium thermocellumsidase produced by Aspergillus oryzae

Okano et al. (2010)

se from Enterobacter cloacae Vasan et al. (2011)se from B. subtilis Zhang et al. (2011a, 2011b)

se from C. thermocellum withe produced by A. oryzae

Tsuchidate et al. (2011)

e from Thermobifida fusca Tanaka et al. (2011)se, cellobiohydrolase, ande from C. cellulolyticum

Ryu and Karim (2011)

e from Endomyces fibuliger, endoglucanasebrio fibrisolvens, cellobiohydrolase frome chrysosporium, and cellodextrinaseaciens

Van Rensburg et al. (1998)

se from T. reesei and β-glucosidase from Fujita et al. (2002)

se and cellobiohydrolase from T. reesei,idase from A. aculeatus

Fujita et al. (2004)

se from T. reesei and β-glucosidase fromopsis fibuligera

Den Haan et al. (2007)

se from A. niger, cellobiohydrolase ande from Thermoascus aurantiacus

Hong et al. (2007)

se and β-glucosidase from A. oryzae Kotaka et al. (2008)se from T. reesei and β-glucosidase from Ito et al. (2009)

se from C. thermocellum, cellobiohydrolase fromum, and β-glucosidase from C. thermocellum

Tsai et al. (2009)

se and cellobiohydrolase from T. reesei, ande from A. aculeatus

Wen et al. (2010)


Yamada et al.(2010a, 2010b, 2010c)

se from T. reesei and β-glucosidase from Yanase et al. (2010a)


Yanase et al. (2010b)

om Streptococcus bovis, glucoamylase fromae, endoglucanase and cellobiohydrolase fromβ-glucosidase from A. aculeatus

Apiwatanapiwat et al. (2011)


Yamada et al. (2011)

se from C. cellulolyticum, and β-glucosidaseocellum

Tsai et al. (2012)



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albus in Z. palmae and Z. mobilis. The resulting strains could produceethanol from cellobiose at more than 95% of the theoretical yield. Vasanet al. (2011) reported the expression of endoglucanase from Enterobactercloacae in Z. mobilis, which could produce 5.5% and 4% (v/v) of ethanolfrom carboxymethyl cellulose (CMC) and NaOH-pretreated bagasse,respectively.

Okano et al. (2010) transferred cellulolytic ability to D-lactic acid-producing Lactobacillus plantarum by expressing endoglucanasefrom Clostridium thermocellum. Although D-lactic acid-producingL. plantarum could assimilate cellooligosaccharide naturally, theresulting endoglucanase-expressing strain produced no detectableamounts of D-lactic acid from a soluble cellulosic material,β-glucan. However, on addition of β-glucosidase into the fermenta-tion medium, the strain could directly produce 1.47 g/L of D-lacticacid with 99.7% optical purity from β-glucan. Tsuchidate et al. (2011)also expressed endoglucanase from C. thermocellum in glutamic acid-producing C. glutamicum. They also added β-glucosidase to the fermen-tationmedium and found that 178 mg/L of glutamic acid was produceddirectly from β-glucan. These were the first reports of direct conversionof cellulosic materials to lactic and glutamic acids.

Zhang et al. (2011a, 2011b) reported overexpression of the endog-enous endoglucanase gene and improved lactic acid-producing abilityin Bacillus subtilis. The B. subtilis strain used in that study naturallyexpressed endoglucanase but its activity was considerably low for as-similation of cellulosic materials as sole carbon sources. However, theendoglucanase-overexpressing and lactic acid productivity-improvedstrain could produce lactic acid directly from amorphous celluloseand some types of pretreated biomass without addition of organicnutrients.

Although there are many reports of production of useful compoundssuch as alcohols, fatty acids, and organic acids through metabolic engi-neering strategies in Escherichia coli, there are only a few reports of con-ferring cellulolytic ability on E. coli. Tanaka et al. (2011) developednovel anchor proteins to display β-glucosidase from Thermobifida fuscaon the surface of E. coli cells using genetic engineering. The recombinantE. coli strain could grow with cellobiose as a sole carbon source. Further-more, Ryu and Karim (2011) successfully displayed endoglucanase,cellobiohydrolase, and β-glucosidase on the E. coli cell surface. They alsoexpressed ethanol-producing enzymes in the resulting strain, whichcould produce ethanol directly from phosphoric acid-swollen cellulose(PASC) and pretreated corn stover.

Cellulose is a large and generally insoluble molecule, and its deg-radation must occur outside cells. However, extracellular productionof cellulase is different from intracellular production of proteins.Although many types of proteins have been produced by E. coli(Salinas et al., 2011; Samuelson, 2011; Waegeman and Soetaert,2011), cellulolytic E. coli has not been reported in the past severalyears, possibly because of its low protein secretion ability (Wood etal., 1997). Because there are many reports of the production ofbio-based alcohols and chemicals from glucose by E. coli via metabolicengineering (Atsumi and Liao, 2008; Zhang et al., 2011a, 2011b), con-struction of an efficient cellulolytic system in E. coli is a promisingstrategy for designing a CBP for producing high-value compoundsfrom lignocellulosic biomass.

2.3. Cellulase expression in yeast strains

Van Rensburg et al. (1998) first reported the expression of multi-ple cellulolytic genes in the ethanol-producing yeast Saccharomycescerevisiae. They developed a cellulolytic S. cerevisiae strain simulta-neously expressing endoglucanase from B. fibrisolvens, cellobiohydrolasefrom Phanerochaete chrysosporium, cellodextrinase from Ruminococcusflavefaciens, and β-glucosidase from Endomycopsis fibuliger. The resultingstrain could grow with cellobiose as a sole carbon source.

Fujita et al. (2002) reported that both endoglucanase from T. reeseiand β-glucosidase from Aspergillus aculeatus were successfully


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expressed in S. cerevisiae for display on its cell surface. The resultingstrain could grow on β-glucan to produce 16.5 g/L of ethanol. Theyalso expressed cellobiohydrolase from T. reesei in cellulolyticS. cerevisiae (Fujita et al., 2004), reporting that the resulting strain pro-duced approximately 3 g/L of ethanol from PASC at 88.5% of the theo-retical yield. There are other reports of expression of three types ofenzymes derived from T. reesei, A. aculeatus, and A. oryzae in S. cerevisiaeto produce ethanol (Ito et al., 2009; Kotaka et al., 2008; Yamada et al.,2010a, 2010b, 2010c, 2011; Yanase et al., 2010b). Apiwatanapiwat etal. (2011) developed cellulose- and amylase-coexpressing S. cerevisiaeto produce ethanol directly and efficiently from cassava pulp containingboth cellulose and starch. In addition, Yamada et al. (2011) reportedthat the cellulolytic ability of recombinant S. cerevisiae improvedwhen diploidization and multi-copy integration of cellulase geneswere combined. The resulting strain could produce 7.5 g/L of ethanolfrom pretreated rice straw, which is an abundant lignocellulosic wastebiomass.

Although S. cerevisiae is inferior to other yeast strains such as Pichiapastoris and Hansenula polymorpha in the amount of protein extracellu-larly secreted (Celik and Calık, 2012), the yeast S. cerevisiae has goodability to secrete proteins than bacterial strains including E. coli (Houet al., 2012; Mattanovich et al., 2012). Accordingly, S. cerevisiae couldbe an ideal host strain to be endowed with cellulolytic ability, becausethe cellulose degradation reaction must occur outside cells. Recently,many types of high-value compounds have been produced by theyeast S. cerevisiae through metabolic engineering (Hong and Nielsen,2012). If the cellulolytic ability of recombinant S. cerevisiae could be im-proved to the level of the cellulose-assimilating fungi T. reesei, it couldbecome a promising host strain for CBP for producing high-value com-pounds from lignocellulosic biomass.

Recently, researchers have given considerable attention to thethermotolerant ethanol-producing yeast Kluyveromyces marxianus,whose use in elevated-temperature fermentation could lower thecost of temperature control and improve cellulose-specific activityusing degradation of lignocellulose in SSF or CBP (Abdel-Banat et al.,2010; Fonseca et al., 2008). The strain can grow and ferment glucoseinto ethanol at temperatures as high as 45 °C, and genetic tools forthe transformation of K. marxianus have already been developed.Hong et al. (2007) reported the expression of endoglucanase fromA. niger and cellobiohydrolase and β-glucosidase from T. aurantiacusin K. marxianus. The resulting strain was able to use cellobiose andCMC as carbon sources and produced 43.4 g/L of ethanol from 10%of cellobiose at 45 °C. Yanase et al. (2010a) reported the successfulexpression of endoglucanase from T. reesei and β-glucosidase fromA. aculeatus on the K. marxinus cell surface. They produced 4.24 g/Lof ethanol from 10 g/L of β-glucan in 12 h at 48 °C. To date most ofthe cellulase genes expressed by yeast strains have been derivedfrom filamentous fungi such as T. reesei, whose optimum growth tem-perature is approximately 50 °C (Hasunuma and Kondo, 2012). Ac-cordingly, K. marxianus, which has the ability to grow and fermentglucose at 45 °C at more than 90% of the theoretical yield, could alsobecome a promising host strain for lignocellulosic CBP at elevatedtemperature.

3. Cellulase overexpression

3.1. Secretion versus cell surface display

Cellulose is a large polymeric and insoluble compound that cannotbe taken up by microbial cells. Thus, for cellulose assimilation, cellu-lase must first be expressed outside the cells, after which cellulosecan be degraded and the glucose product taken up by the cell. Twomodes of expression of an enzyme outside cells may be considered:secretion and cell surface display (Figs. 2 and 3). The efficientcellulose-assimilating filamentous fungi T. reesei secretes more than10 types of cellulases outside the cells, with amounts of cellulases



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Fig. 2. Schematic illustration of cellulose assimilation by cellulase-secreting microorganisms.


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reported to reach as high as several dozen g/L (Peterson andNevalainen, 2012). In contrast, efficient cellulose-assimilating anaer-obic clostridial bacteria display on their cell surfaces a cellulase com-plex called the cellulosome that contains many types of cellulases(Schwarz, 2001; Tamaru et al., 2010). In the surface-display system,the amount of cellulase displayed is strictly dependent on the cell sur-face area. In contrast, in secretion, because there are no such limitsoutside the cell, high levels of cellulase expression may be expected.Another disadvantage of the surface-display system is the low diffu-sion rate of cellulase immobilized on the cell surface, owing to its in-solubility in the substrate. Although a high diffusion rate is desirablefor cellulase, cell surface-displayed enzymes show lower diffusionrates than secreted enzymes (Khaw et al., 2006). But there are someadvantages to displaying cellulases on the cell surface. As described

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Fig. 3. Schematic illustration of cellulose assimilation


ED Pin previous sections, cellulose is degraded by a synergistic action of

three types of cellulases. In surface display, these three types of en-zymes are located in close proximity, and this proximity could im-prove their synergism. In fact, the specific activity of cellulosome, inwhich cellulases are located in close proximity, was reported to be50 times higher than that of the cellulases secreted by T. reesei, inwhich cellulase are diffused (Schwarz, 2001). In producing ethanolfrom hydrothermally pretreated rice straw, the display of cellulaseson the yeast cell surface allowed a reduction in the amount of com-mercial cellulase added to SSF (Matano et al., 2012a). Enhanced yieldsof ethanol produced from cellulose would be achieved by the allevia-tion of irreversible desorption of cellulase on crystalline cellulose(Matano et al., 2012b). Another advantage of surface display is recy-clability of the enzyme. Surface-displayed cellulase can be recovered

by cellulase surface-displaying microorganisms.



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and reused by sedimentation or centrifugation. In addition, becauseβ-glucosidase, which degrades cellooligosaccharide into glucose, isalso displayed on the cell surface, glucose is released very close tothe cell surface and may be taken up immediately by cells, in contrastto β-glucosidase secreting strains (Tsai et al., 2012). By this meansglucose concentration in the medium may be maintained at a lowlevel, reducing the risk of contamination. This advantage would be es-pecially useful for long-term, large-scale industrial applications.

There are also two means of expressing cellulase outside the cellsin recombinant cellulolytic microorganisms. There are reports ofcellulase-secreting bacterial strains including K. oxytoca, Z. mobilis,Z. palmae, L. plantarum, B. subtilis, C. glutamicum (Okano et al., 2010;Tsuchidate et al., 2011; Vasan et al., 2011; Yanase et al., 2005a,2005b; Zhang et al., 2011a, 2011b; Zhou and Ingram, 2001; Zhou etal., 2001). In some cases, signal sequence for secretion could be an im-portant issue, because it strongly affects secretion efficiency and lo-calization of cellulase (Tsuchidate et al., 2011; Yanase et al., 2005a;Zhang et al., 2011a, 2011b). A cellulase surface-displaying E. coli strainhas been also developed. Tanaka et al. (2011) developed a novel an-chor protein named Blc to display β-glucosidase from T. fusca, andthe β-glucosidase-displaying strain was able to use cellobiose as itssole carbon source. Ryu and Karim (2011) reported the display ofendoglucanase, cellobiohydrolase, and β-glucosidase on the cell sur-face of E. coli using a PgsA anchor, and the resulting strain could fer-ment PASC and pretreated corn stover directly into ethanol. In abacterial cellulolytic system, the expression of cellulase outside thecells in an active form is a limitation. Thus, for the construction of ef-ficient cellulolytic bacteria, selection of a suitable cellulase gene, sig-nal sequence, and mode of expression are important.

Similarly in bacteria, there are two modes of expression of cellulasein recombinant cellulolytic S. cerevisiae and K. marxianus. The native se-cretion signal of cellulase is commonly replaced with the secretion sig-nal of α-factor derived from S. cerevisiae (Hou et al., 2012). Thesecretion signal of glucoamylase derived from Rhizopus oryzae is alsoused for the secretion of cellulase (Yanase et al., 2010b). Hong et al.(2007) successfully secreted endoglucanase, cellobiohydrolase, andβ-glucosidase using the secretion signal of α-factor derived fromS. cerevisiae. To display cellulase on yeast cell surfaces, α-agglutinin iscommonly used as the anchor protein. Fujita et al. (2002, 2004) werethe first to succeed in displaying cellulase on the S. cerevisiae cell surfaceand fermenting β-glucan and PASC directly into ethanol. Yanase et al.(2010a) developed a cellulose surface-displaying K. marxianus strainusing anα-agglutinin anchor protein derived from S. cerevisiae and pro-duced ethanol directly from β-glucan at elevated temperatures.

As mentioned above, there are advantages and disadvantages ofsecretion and surface display. Yanase et al. (2010b) compared theethanol productivity of a cellulase-secreting strain and a cellulase-displaying strain in the fermentation of β-glucan with S. cerevisiae.The strains produced 1.6 and 2.1 g/L of ethanol, respectively. Theseauthors concluded that to construct cellulolytic S. cerevisiae, surfacedisplay is superior to secretion. However, this situation could varydepending on many factors such as the host strain, total amount ofexpressed cellulase, and fermentation conditions. A suitable expres-sion mode should be selected to design an efficient CBP using cellulo-lytic microorganisms.

A strategy aimed at regulating and optimizing cellulase ratios iscalled the minicellulosome strategy (Fig. 4) (Ito et al., 2009; Tsai et al.,2009, 2012; Wen et al., 2010). Ito et al. (2009) reported the display oftwo Z domains of protein A derived from Staphylococcus aureus andtwo cohesins derived from C. cellulovorans on the cell surface of S.cerevisiae via an α-agglutinin anchor protein. In addition, they showedthe simultaneous expression of β-glucosidase fused with dockerin,which has a strong interaction with cohesin, and endoglucanase fusedwith the Fc domain of human IgG, which has a strong interaction withthe Z domain. The resulting strain displayed the β-glucosidase andendoglucanase in a 1:1 ratio. The authors indicated that the use of this


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methodwould allowdisplaying enzymes in the desired ratios. Other re-search groups then developed different types of enzyme-expression-ratio-regulated systems in S. cerevisiae using a-agglutinin as an anchorprotein (Tsai et al., 2009, 2012; Wen et al., 2010).

Use of these strategies allows the regulation of cellulase expressionratios. In this manner, recombinant cellulolytic technology may ap-proach the T. reesei cellulase expression system. However, thesemethods are not optimized for the environment but for regulation. Ex-pression regulation methods could be used for optimization if the opti-mum ratios were obvious. However, in most cases the optimumcellulase ratio for efficient degradation of lignocellulose is unknown.Yamada et al. (2010a, 2010b, 2010c) reported a cellulose-expression-ratio-optimizing method. They constructed a S. cerevisiae library ex-pressing various cellulose-displaying ratios, and then identified acellulose-expression-ratio-optimized strain by screening for growthability on target cellulosic material. This strain carried the expectedcopy number of cellulase genes and showed high cellulolytic activitycompared to a classical cellulase-overproducing strain. Using this strat-egy the cellulase expression ratio could be optimized for degradation oftarget cellulosic materials.

3.2. Overexpression strategies of cellulase

Thehigh cellulose assimilation ability of T. reesei is because of its highcellulase-producing ability as well as its regulation of the expression ra-tios of many types of cellulases depending on environmental substrateconditions (Peterson and Nevalainen, 2012; Stricker et al., 2008). Ifthese abilities could be conferred on recombinant cellulolytic microor-ganisms, efficient cellulolytic microorganisms could be constructed.

There are many reports of overproduction of enzymes using tech-niques such as promoter engineering, enhancement of gene copynumber, and protease deficiency. A promoter-engineering strategyaims to enhance transcription efficiency by tailoring the promoter se-quence to produce large amounts of an enzyme. To control transcrip-tion efficiency, artificial promoters have been developed in yeast andbacterial strains (Alper et al., 2005; Jensen and Hammer, 1998;Nevoigt et al., 2006). Nevoigt et al. (2006) constructed a random mu-tant library of the strong yeast constitutive promoter TEF1 using anerror-prone PCR method. They screened 11 mutants, which showedvarious reporter enzyme activities ranging from 8% to 120% of thatof the parental promoter. By promoter engineering, the transcriptionefficiency, which is the efficiency of the first step of protein produc-tion, can be improved.

Another strategy for improving transcription activity is the enhance-ment of gene copy number. Use of episome recombination allows en-hancing the expression level of target genes by increasing their copynumber to high levels. However, the stability of episome DNA is gener-ally lower than that of integrated DNA in the absence of selective pres-sure, therefore the use of episome recombination would be difficult inindustrial applications. On the other hand, although integrative geno-mic recombination is more stable than episome recombination, its tar-get gene copy number and thus the gene expression level are alsovery low. To overcome these disadvantages, a multi-copy integrationmethod has been developed (Lopes et al., 1991; Sakai et al., 1990).This method results in both high transcription activity and gene stabil-ity. Recently, a novel technique for stably enhancing target gene copynumbers in S. cerevisiae has been developed (Yamada et al., 2010a,2011). Yamada et al. constructed a multi-copy gene of endoglucanase,cellobiohydrolase, andβ-glucosidase integrated into the S. cerevisiae ge-nome. The resulting strain was mated to integrate the multi-copy geneinto a diploid strain. This method drastically increased the cellulolyticactivity of the strain, which produced 7.6 and 7.5 g/L of ethanol directlyfrom PASC and pretreated rice straw, respectively. The correlation be-tween gene copy number and level of protein expression has beenreviewed previously (Yamada et al., 2010b). A strategy to improve tran-scription activity would be effective when transcription becomes the



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Fig. 4. Schematic illustration of cellulose assimilation by minicellulosome-expressing microorganisms.


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rate-limiting step. However, overenhancement of transcription activitywill result in a shortage of transcription factors (Valkonen et al., 2003).So achieving efficient overproduction of cellulose will depend on theidentification and improvement of the rates of rate-limiting steps in-cluding transcription, translation, posttranslational modification, andsecretion.

The degradation of a target protein by proteases derived from thehost strain may be an important issue. In general, microorganismspossess native protease activities used for maintaining their own lifeactivities and harmless to their own cells. However, a heterogeneousprotein may be degraded by a protease produced by a host strain, andconsequently, produce inadequate amounts of protein or lose activity.To overcome this problem, a protease-gene-deficiency strategy hasbeen developed. Protease genes have been deleted in species suchas B. subtilis, S. cerevisiae, A. oryzae, and P. pastoris (Cereghino andCregg, 2000; Sander et al., 1994; van den Hombergh et al., 1997;Wu et al., 1991). Wu et al. (1991) reported that the deletion of six ex-tracellular protease genes in B. subtilis cause the extracellular prote-ase to decrease to 0.32% of that in the parental strain. The stabilityof a heterologous protein expressed by the strain was drastically im-proved. However, the native protease is expressed to maintain the lifeactivity of the host strain, so that protease deficiency could reduceits survival and growth rates (Cereghino and Cregg, 2000). InS. cerevisiae, secreted proteins are often modified by yeast-specifichypermannosylation, which may affect enzyme activities assembledon the yeast cell surface. Recently, Suzuki et al. (2012) reporteddeglycosylation of a minicellulosome displayed on the surface ofS. cerevisiae using glycosylation mutants. The recombinant strainshowed enhanced cellulosome assembly on the yeast cell surface.

As described in this section, cellulase expression ratio regulationand optimization, which are important abilities of the efficientcellulose-assimilating fungi T. reesei, could be achieved in recombi-nant cellulolytic microorganisms. However, amount of secreted cellu-lase in recombinant cellulolytic microorganisms remained aroundseveral dozen mg/L and its cellulase activity remained around severalhundred filter paper unit (FPU)/L (Vasan et al., 2011; Zhang et al.,2011a, 2011b), and it is 100 to 1000 fold lower than T. reesei cellulase


EDsystem, which reaches several dozen g/L and tens of thousands FPU/L

(Peterson and Nevalainen, 2012). Thus, if cellulase expression optimi-zation and cellulase overproduction reaching several dozen g/L areachieved simultaneously in future work, an industrial CBP for produc-ing high-value compounds from lignocellulosic biomass using recom-binant cellulolytic microorganisms may be realized.

4. Molecular engineering of cellulases

Many efforts have been made to improve specific cellulase proper-ties by genetic engineering. Three major strategies are generally usedto engineer cellulase for increased hydrolytic potential: (1) rationaldesign, (2) directed evolution, and (3) protein fusion.

Rational design is the earliest strategy for cellulase engineering andis based on the knowledge of the structure–function relationship be-tween cellulase and crystalline cellulose. Identification of catalyticsites in enzymes, usually based on crystallographic structure, can leadto the development of new catalytic mechanisms that confer improvedhydrolytic activity on cellulase via the substitution of amino acids in-volved in the reaction by genetic engineering (Baker et al., 2005;Dwyer et al., 2004). The structural stability of cellulase can be estimatedby SCHEMA, which is a computational approach to identify blocks of se-quences that minimize structural disruption of proteins. Heinzelman etal. (2009a, b) described the quantitative prediction of cellulase thermo-stability and identification of stabilizedmutants. Mutations designed bythe SCHEMA approach stabilized and improved Avicel hydrolysis byCBH2 derived from various fungi such as Humicola insolens, Hypocreajecorina (anamorph T. reesei), and P. chrysosporium (Heinzelman et al.,2009a). Thus, rational design is a logical method to engineer a proteinby substituting amino acids. However, successful computationalmodelsdepend on knowledge of the biochemical mechanisms involved in cel-lulose hydrolysis, often limiting the creation of higher-activity mutants(Percival Zhang et al., 2006).

As an alternative to cellulase engineering, directed evolution al-lows improvement in specific activities of enzymes. This strategycan improve enzymes of interest through iterative cycles of randommutagenesis and screening. By selection of advantageous mutations



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that afford more beneficial phenotypes than the wild type, not onlyenhanced cellulolytic activity but improved enzyme stability at abroad range of pH and temperature have been implemented in cellu-lase (reviewed in Percival Zhang et al., 2006). DNA shuffling anderror-prone PCR have been applied as mutagenesis techniques toalter the properties of cellulases including endoglucanase and BGL(Kim et al., 2000; McCarthy et al., 2004; Murashima et al., 2002).Liang et al. (2011) reported the directed evolution of a thermophilicendoglucanase (Cel5A) from Thermoanaerobacter tengcongensis.After three rounds of error-prone PCR and Congo red-based screeningof 4700 mutants, Cel5A variants showed almost twofold higher CMChydrolysis activity than the wild type, owing to reduced number ofintra-Cel5A hydrogen bonds. Most recently, Ito et al. (2012) identifiedadvantageous mutations that conferred thermostability on CBH2derived from the mesophilic fungus P. chrysosporium, using a mutantlibrary constructed based on the consensus sequence of variousthermophilic fungal CBH2s. To improve the thermostability ofP. chrysosporium CBH2, the advantageous mutations were accumulatedwithin the wild-type genome. The thermostable variant of CBH2obtained in the experiment retained sufficient activity at 50 °C formore than 72 h (Ito et al., 2012). Random approaches coupled with en-zyme in vitro evolution have beenused for the improvement of thermo-stability (Ruller et al., 2008; Stephens et al., 2007) and alkaliphilicity(Inami et al., 2003; Stephens et al., 2009) of hemicellulolytic enzymesas well as cellulases. Song et al. (2012) improved the hydrolytic perfor-mance of a GH11 xylanase from Thermobacillus xylanilyticus using acombination of random mutagenesis and gene shuffling. Several vari-ants showed increased hydrolytic activity on wheat straw and im-proved synergistic action with a commercial cellulase reagent.

In directed evolution, screening is a critical step for finding desiredmutants. Endoglucanase activities are often estimated by halo forma-tion on a solid agar plate containing a soluble cellulosic substrate suchas CMC or PASC. Activities of BGL are spectrophotometrically deter-mined with chromogenic substrates. However, abilities of crystallinecellulose hydrolysis cannot be evaluated with these soluble sub-strates, making selection of a desired CBH difficult. Developing thebest screening method for mutants effectively hydrolyzing lignocellu-losic materials requires the use of insoluble celluloses as substrates.

Since crystalline cellulose is hydrolyzed by the synergetic reaction ofcellulases including endoglucanase, cellobiohydrolase, and BGL toproduce glucose as mentioned above, combining cellulases as fusionproteins is a promising strategy for improving the efficiency of cellulosehydrolysis, owing to the proximity effects of the fused enzymes. Artificialfusion proteins consisting of endoglucanase and cellobiohydrolase havebeen constructed using genes of Cellulomonas fimi and C. stercorarium(Riedel and Bronnenmeier, 1998;Warren et al., 1987). Recently, a fusionof β-glucosidase (CcBG) from C. cellulovorans with a cellulosomalendoglucanase (CtCD) from C. thermocellum resulted in twofold higherglucose production from PASC and threefold lower cellobiose accumula-tion than a mixture of the single enzymes. The CtCD–CcBG bifunctionalenzyme also improved thermostability with a melting point of 10.9 °Chigher than that of CcBG (Lee et al., 2011). Adlakha et al. (2011) synthe-sized recombinant bifunctional enzymes based on β-1,4-endoglucanase(Endo5A) and β-1,4-endoxynalase (Xyl1D) from a Panibacillus strainisolated from the gut of a cotton bollworm. The chimeric Endo5A-Xyl1D protein interconnected by a glycine–serine linker, expressed inE. coli, demonstrated 1.6-fold and 2.3-fold higher hydrolysis activitythan Endo5A and Xyl11D, respectively. Zou et al. (2012) constructed afusion gene of cbh1 from T. reesei and e1 encoding an endoglucanasefrom Acidothermus cellulolyticus with a flexible polyglycine linker andrigid α-helix linker, expressing the fusion in T. reesei under the controlof amodified cbh1promoter. The recombinant T. reesei strain showed in-creases of 40% at 55 °C and 169% at 60 °C in the hydrolysis of pretreatedcorn stover compared to its parent strain. Improved thermostability ofthe enzymewas conferred by connecting the thermostableAcidothermuscellulolyticus endoglucanase to T. reesei CBH1.


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5. Conclusion

In 1998, Van Rensburg et al. first reported a cellulolytic yeast able toassimilate cellobiose as its sole carbon source. Over the past years, tech-nology for constructing cellulolytic microorganisms has been well de-veloped. Bacterial cellulolytic strains of K. oxytoca, Z. mobilis, Z. palmae,L. plantarum, B. subtilis, C. glutamicum, and E. coli have been developed.The yeast strains S. cerevisiae and K. marxianus have been used for ex-pressing cellulase by secretion and display. Many types of cellulasessuch as endoglucanase, cellobiohydrolase, and β-glucosidase havebeen successfully expressed, and many types of cellulosic materialssuch as CMC, β-glucan, PASC, and pretreated biomass can now be as-similated and fermented by recombinant cellulolytic microorganisms.Furthermore, the technology for improving cellulose-specific activityby rational design, directed evolution, and protein fusion has beenwell developed. However, to date, commercial CBP from lignocellulosehas not been established yet because of many problems involved inthe development of CBP microorganisms. For example, cellulolyticactivity of recombinant cellulose-hydrolyzing microorganisms is com-parably lower than that of cellulolytic fungi such as T. reesei. Further-more, the microorganisms should have high robustness assimilatingmany kinds of monosaccharide and tolerating many kinds of inhibitorycompounds derived from lignocellulosic biomass. Besides, efficient andcost-effective pretreatment method for lignocellulosic biomass has notalso been established yet. In future research, improvement in amountsof expressed cellulase, development of novel cellulases with high spe-cific activity, conferring high robustness for CBP microorganisms, anddevelopment of efficient and cost-effective pretreatment method,should be emphasized for commercialization of CBP from lignocellulos-ic biomass.

Recently, many types of biofuels and chemicals have been producedfrom glucose by metabolic engineered microorganisms (Atsumi andLiao, 2008; Hong and Nielsen, 2012; Zhang et al., 2011a, 2011b). If cel-lulolytic microorganisms with sufficient cellulolytic activity could bedeveloped, an industrial CBP for producing high-value compoundsfrom lignocellulosic biomass would become a promisingmeans of real-izing a society based on sustainable material cycling.

Acknowledgments

This work was supported by Special Coordination Funds for Pro-moting Science and Technology, Creation of Innovation Centers forAdvanced Interdisciplinary Research Areas (Innovative BioproductionKobe), MEXT, Japan.

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