Engineering cellulolytic ability into bioprocessing organisms - CiteSeerX

Appl Microbiol Biotechnol (2010) 87:1195–1208 DOI 10.1007/s00253-010-2660-x

MINI-REVIEW

Engineering cellulolytic ability into bioprocessing organisms Daniel C. la Grange & Riaan den Haan & Willem H. van Zyl

Received: 1 March 2010 / Revised: 2 May 2010 / Accepted: 2 May 2010 / Published online: 28 May 2010 # Springer-Verlag 2010

Abstract Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates. Keywords Cellulases . Recombinant microorganisms . Lignocellulosic biomass . Consolidated bioprocessing

D. C. la Grange : R. den Haan : W. H. van Zyl (*) Department of Microbiology, University of Stellenbosch, De Beer Street, Stellenbosch 7600, South Africa e-mail: [email protected]

Introduction With a demand of more than 84 million barrels per day (30 billion per year), it is safe to say that the world is currently heavily dependent on oil, especially in the transport sector (Energy Information Administration 2005). However, rising oil prices, concern about environmental impact, and supply instability are among the factors that have lead to greater interest in renewable fuel and green chemistry alternatives. Biofuels should ideally retain the advantages of oil with regards to being relatively cheap and rich in energy and should in addition provide a net energy gain, have environmental benefits, and be producible in large quantities without impacting on food supplies (Hill et al. 2006). Plant biomass is therefore the only foreseeable renewable feedstock for sustainable production of biofuels. The main technological impediment to more widespread utilization of this resource for production of fuels and chemicals is the lack of low-cost technologies to overcome the recalcitrance of the cellulosic structure (Van Zyl et al. 2007). Producing biofuels such as ethanol from cellulosic plant material has the potential to meet capacity requirements without impacting directly on food production (Maly and Degen 2001). Lignocellulosic plant biomass represents the largest source of renewable carbon and consists of 40–55% cellulose, 25–50% hemicellulose and 10–40% lignin, depending on whether the source is hardwood, softwood, or grasses (Sun and Cheng 2002). The exact composition therefore differs significantly between different plant sources. The major polysaccharide present is waterinsoluble cellulose that contains the major fraction of fermentable sugars. Full enzymatic hydrolysis of crystalline cellulose requires synergistic action of three major types of enzymatic activity (1) endoglucanases (EGs) (1,4-β-D-glucan 4-glucanohydrolases; EC 3.2.1.4), (2) exoglucanases, including cellodextrinases (1,4-β-D-

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glucan glucanohydrolases; EC 3.2.1.74), and cellobiohydrolases (CBHs) (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91), and (3) β-glucosidases (BGL) (β-glucoside glucohydrolases; EC 3.2.1.21) (Zhang and Lynd 2004). Endoglucanases are active on the amorphous regions of cellulose and yield cellobiose and cello-oligosaccharides as hydrolysis products. Cellobiohydrolases are active on the crystalline regions of cellulose and yield almost exclusively cellobiose as their main hydrolysis product. In turn, β-glucosidases convert cellobiose and some cellooligosaccharides to glucose. The hemicellulose fraction generally consists of (arabino)xylan and galacto(gluco) mannan (Sun and Cheng 2002). The hydrolysis of this fraction requires the synergistic action of a plethora of enzymes for complete hydrolysis. While several microorganisms can be found in nature with the ability to produce the required enzymes to hydrolyse all the polysaccharides found in lignocellulose, one of the main technical challenges in converting lignocellulosic biomass to commodity products is that there is no organism with the ability to directly hydrolyze these polysaccharides and ferment the liberated sugars to a desired product such as ethanol, butanol, or lactic acid at rates and titers required for economic feasibility. Current technologies for conversion of biomass commences with a pretreatment step during which physical and/or chemical processes are used to render the polymeric sugar fractions more accessible to conversion by enzymatic processes (Stephanopoulos 2007). The type of pretreatment defines the optimal enzyme mixture to be used and the composition of the hydrolysis products. Subsequent biological conversion typically involves four steps: the production of enzymes (cellulases and hemicellulases), the hydrolysis of cellulose and hemicelluloses to sugars, and the fermentation of hexose sugars and pentose sugars to commodity products (Lynd et al. 2005). Improvements of biomass conversion technology generally entail the consolidation of two or more of these steps. Consolidated bioprocessing (CBP), where all four steps are combined in a single reactor, has the potential to greatly reduce processing cost compared to separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and simultaneous saccharification and co-fermentation (SSCF). There have been reports showing conversion of biomass to ethanol in an SSF process using a co-culture of the yeasts Saccharomyces cerevisiae and Brettanomyces clausenii on laboratory scale (Spindler et al. 1989). It may therefore be possible to perform CBP with a mixture of organisms having the desired properties of cellulolytic ability and product formation. However, the use of mixed cultures on industrial scale is likely to be problematic due to problems such as difficulties in maintaining the optimal ratios of the organisms, incompatible optimal conditions of the organisms involved, and

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differing susceptibilities to inhibitors present in lignocellulosic hydrolysates. Therefore, the key to CBP is the engineering of a microorganism that can efficiently de-polymerize biomass polysaccharides to fermentable sugars and efficiently ferment this mixed-sugar hydrolysate (Hahn-Hagerdal et al. 2006). Due to the variety of feedstocks likely to be used, the diversity in pretreatment methods and the difference in desired products produced, there is scope for development of organisms with a range of different traits. The high-solid environment likely to be required for an economically feasible industrial cellulose conversion process will also require the organism to be robust with regards to inhibitor tolerance. Furthermore, characteristics such as the ability to simultaneously utilize sugars, GRAS status, minimal nutrient supplementation, and tolerance of low pH and high temperature would also be desirable in a CBP organism (Van Zyl et al. 2007; Zaldivar et al. 2001). The development of such an organism has focused on one of two strategies (Lynd et al. 2005). The native cellulolytic strategy involves engineering naturally cellulolytic microorganisms to improve product-related properties, such as yield. The recombinant cellulolytic strategy involves engineering non-cellulolytic organisms with high product yields so that they express a heterologous cellulase system to enable cellulose utilization. While there are several promising cellulolytic or partially cellulolytic organisms such as Clostridium thermocellum (Lynd et al. 2005) and Geobacillus (Cripps et al. 2009) that are being manipulated for use in industrial biomass conversion, this paper will review the engineering of microorganisms with interesting product-forming abilities or other inherent advantages to break down cellulose. The variety of yeasts and bacteria discussed in the following sections all have some innate or engineered advantage such as substrate range, process hardiness, pH and/or thermotolerance, inhibitor tolerance, or product formation ability that would make it useful in biomass conversion. The expression of cellulase-encoding genes within these organisms to enable growth on cellulosic substrates is discussed.

Engineering eukaryotic organisms to hydrolyze polysaccharides Saccharomyces cerevisiae The use of S. cerevisiae for the production of ethanol from hexose sugars (particularly glucose) is well established in industry (Kuyper et al. 2004; Nissen et al. 2000; Van Dijken et al. 2000). S. cerevisiae has many positive attributes, which makes it suitable for industrial use. It can produce ethanol at a high rate (3.3 g/L/h) from glucose,

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and it can grow at a low pH. Furthermore, S. cerevisiae is generally regarded as safe, and many strains are known to be robust in industrial applications. However, this yeast species also has a number of shortcomings that need to be addressed before it can be considered as a process organism for CBP. The main inadequacies are the yeast's inability to hydrolyze polysaccharides (cellulose and hemicellulose); that it cannot grow on or produce ethanol from xylose or arabinose, and that it is not able to tolerate high temperatures. A number of research groups around the world have been working on improving the substrate range of S. cerevisiae to include the sugars contained in plant biomass (Hahn-Hagerdal et al. 2001, 2007; Karhumaa et al. 2006; Kuyper et al. 2004; Van Rooyen et al. 2005). A S. cerevisiae strain that expressed the xylose isomerase gene from the fungus Piromyces sp. E2 was further metabolically engineered to allow anaerobic growth on xylose in synthetic media with a maximum specific growth rate μmax of 0.09 h−1 (Kuyper et al. 2004). During growth on xylose, this strain did not produce xylulose, and xylitol production was negligible. Laboratory and industrial S. cerevisiae strains were engineered to co-ferment the pentose sugars D-xylose and L-arabinose (Karhumaa et al. 2006). Introduction of a fungal xylose and a bacterial arabinose utilization pathway resulted in strains able to grow on both of these pentose sugars. However, the L-arabinose reductase activity of the xylose reductase resulted in conversion of arabinose into arabitol. The engineered industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose, and simultaneous co-utilization of xylose and arabinose could be demonstrated. There have therefore been several instances of successful engineering of S. cerevisiae strains to utilize soluble sugars. While high rate utilization of pentose sugars as well as co-utilization of pentose and hexose sugars remains a significant challenge, a potential CBP organism also needs to utilize the insoluble sugars present in lignocellulosic biomass. There have been a large number of reports detailing the expression of one or more cellulase-encoding genes in S. cerevisiae (see Van Zyl et al. (2007) for a review). Strains of S. cerevisiae were created that could grow on and ferment cellobiose, the main product of the action of cellobiohydrolases on cellulosic substrates. Expression of the Saccharomycopsis fibuligera β-glucosidase-encoding gene enabled a recombinant S. cerevisiae strain to grow on cellobiose at approximately the same rate as on glucose in anaerobic conditions (Van Rooyen et al. 2005). A number of studies have shown co-production of cellulases specifically with the aim of enabling the organism to grow on a polymeric substrate. Van Rensburg et al. (1998) constructed a yeast strain capable of hydrolyzing numerous cellulosic substrates and growing on cellobiose. Cho et al. (1999) showed that for SSF experiments with their strain producing a β-glucosidase

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and an enzyme with both exo- and endocellulase activity, loadings of externally added cellulase could be decreased. Fujita et al. (2002, 2003) reported co-expression and surface display of cellulases in S. cerevisiae. A recombinant strain simultaneously displaying the Trichoderma reesei endoglucanase II, cellobiohydrolase II, and the Aspergillus aculeatus β-glucosidase was created. High cell density suspensions of this strain were able to directly convert phosphoric acid swollen cellulose (PASC) to ethanol with a yield of approximately 3 g/L from 10 g/L PASC within 40 h (Fujita et al. 2003). However, growth of this strain on the cellulosic substrate was not demonstrated. Den Haan et al. (2007b) reported growth on and direct conversion of PASC to ethanol by an S. cerevisiae strain co-expressing the T. reesei endoglucanase 1 (cel7B) and the S. fibuligera β-glucosidase 1 (bgl3A). Anaerobic growth at a rate of ∼0.03 h−1 up to levels of ∼0.27 g/L dry cell weight was observed with this strain on medium-containing yeast extract, peptone and 10 g/L PASC as sole carbohydrate source with concomitant ethanol production of up to 1.0 g/L. Jeon et al. (2009) recently constructed a similar strain expressing the S. fibuligera bgl3A and the C. thermocellum cel5E endoglucanase genes. This strain produced significantly more endoglucanase activity than the one reported by Den Haan et al. (2007b), and notably improved conversion of PASC to ethanol was achieved. As exoglucanase activity such as CBH is required for the successful hydrolysis of crystalline cellulose, it is hypothesized that the addition of successful, high-level expression of a CBH(s) to these strains will enable conversion of crystalline cellulose to ethanol. However, despite several reports of successful expression of CBH-encoding genes in S. cerevisiae, the titres achieved were generally too low to allow CBP (Den Haan et al. 2007a). The challenge in this field will be to alleviate this problem through a combination of finding gene candidates that are compatible with expression in yeast, improving specific activities of the enzymes and metabolic engineering of the yeast host. Another way of engineering cellulose hydrolysis in non-cellulolytic organisms could be by producing a cellulosome. Most of the cellulases expressed in S. cerevisiae to date are non-complexed or free enzymes; however, the most effective cellulolytic organisms make use of complexed enzymes (Devaux 2004; Doi 2008; Doi et al. 1998; Doi et al. 2003; Fierobe et al. 2008; Maki et al. 2009). In their search for the molecular component on the cell surface of C. thermocellum, responsible for specific binding to cellulose (cellulose-binding factor), Lamed et al. (1983a, b) discovered and defined the cellulosome (Bayer et al. 2008). Cellulosomes are extracellular multienzyme systems produced by cellulolytic bacteria to degrade crystalline cellulose. These multienzyme complexes typically consist of a scaffoldin molecule with enzymatic units attached to it.

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Scaffoldins contain cohesin domains to which enzymatic units can bind by means of their respective dockerin domains. The cohesin–dockerin interaction is Ca2+-dependent, and binding is species-specific. Cellulases from C. thermocellum failed to interact with the scaffoldin protein from C. cellulolyticum and vice versa (Fierobe et al. 1999). In cells growing on cellulose, cellulosomes are typically attached to the host cell surface and they also contain at least one cellulose-binding domain enabling the whole complex to effectively bind to cellulose. This arrangement enables enzyme proximity synergy as well as enzyme–substrate– microbe synergy; as a result, cellulosomes are much more efficient in breaking down cellulose than free enzymes. Furthermore, the species specificity of cohesin–dockerin binding allows the design of mini-cellulosomes to contain enzymes of interest (Fierobe et al. 2005, 2008). Tsai et al. (2009) demonstrated the functional assembly of a mini-cellulosome on the surface of S. cerevisiae. The scaffoldin contained three divergent cohesin domains from C. thermocellum, Clostridium cellulyticum, and Ruminococcus flavifaciens and was attached to the cell surface of S. cerevisiae using the glycosylphophatidylinositol (GPI) anchor. Incubation with E. coli lysates containing an endoglucanase, an exoglucanase, and a β-glucosidase, each with an appropriate dockerin resulted in the assembly of a functional mini-cellulosome on the yeast cell surface. Compared with equivalent amounts of free enzyme, the mini-cellulosome displayed enhanced glucose liberation and host ethanol production on PASC. The final ethanol concentration was 3.5 g/L; this was 2.6-fold higher than that obtained with the free enzymes. This demonstrated the potential of developing a functional S. cerevisiae cellulosome. Wen et al. (2010) constructed a scaffoldin containing three C. thermocellum cohesins as well as the C. thermocellum cellulose-binding domain. The S. cerevisiae αagglutinin anchor was used to tether the scaffoldin to the yeast cell surface. C. thermocellum dockerins were added to a T. reesei Cel5A (EGII) and Cel6A (CBHII) as well as the A. aculeatus β-glucosidase. The scaffoldin and the three dockerin-containing cellulases were expressed from two episomal plasmids in S. cerevisiae. Although ethanol production was low, the authors were able to show enzyme–enzyme as well as proximity synergy. Kluyveromyces marxianus Processing biomass to produce commodity products inevitably starts with a pretreatment step (Lynd et al. 2002). This usually involves a heat intensive method; after which, the biomass feedstock needs to be cooled down to a temperature at which subsequently added enzymes and a fermentation organism can function. If the commodity product to be produced is ethanol, the mixture then has to

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be reheated for the distillation process. It would therefore be advantageous if the biologically mediated processing steps could occur at an elevated temperature as it would increase enzyme activity and decrease the amount of cooling required, thereby decreasing cost, and it would furthermore reduce the risk of contamination. It is for this reason that there is a lot of interest in developing cellulolytic thermophiles as CBP organisms and also in developing thermostable enzymes for this industry (Lynd et al. 2005). Several industrial S. cerevisiae strains can grow at 35 °C and even up to 40 °C. However, some strains of the yeast K. marxianus can grow at temperatures as high as 52 °C and have a short generation time and high growth rate at elevated temperatures (Rajoka et al. 2003). K. marxianus can utilize a vast range of substrates, including xylose to produce ethanol (Fonseca et al. 2007, 2008). Successful SSF with a variety of feedstocks at elevated temperatures was demonstrated with K. marxianus. This yeast is therefore an interesting candidate for CBP because of its ability to grow at high temperatures with short doubling times on inexpensive substrates. The cellobiohydrolase 1 (cel7A), endoglucanase 1 (cel5A) and β-glucosidase (bgl3A) genes from the thermophilic fungus Thermoascus aurantiacus were all cloned and expressed in various yeasts (Hong et al. 2003a, b; 2007a). The recombinant cellulase enzymes were all shown to be thermotolerant. Subsequently, the same research group combined thermotolerant cellulase activities in a strain of K. marxianus (Hong et al. 2007b). The resulting strain was able to grow in synthetic media containing cellobiose or carboxymethylcellulose (CMC) as sole carbon source. Furthermore, the strain could ferment 100 g/L cellobiose to 43.4 g/L ethanol in 24 h at 45 °C. Hydrolysis of crystalline cellulose was not shown. Pichia stipitis Pichia stipitis was isolated from decaying wood and the larvae of wood-inhabiting insects (Jeffries et al. 1996). It is one of the best studied xylose-fermenting yeasts. P. stipitis strains produce low quantities of various cellulases and hemicellulases to break down wood into monomeric sugars although it cannot utilize polymeric cellulose as carbon source (Jeffries et al. 2007). Among the enzymes that are naturally produced are β-glucosidase that allows the yeast to ferment cellobiose. Furthermore P. stipitis is also able to ferment glucose, xylose, mannose, and galactose (Parekh and Wayman 1986). It furthermore has the ability to produce cell mass from L-arabinose but not ethanol (Nigam 2002). The enzymes for xylose metabolism in P. stipitis are induced by xylose and repressed by glucose. Fermentation results on P. stipitis show that 61 g ethanol/L can be produced in synthetic media (Slininger et al. 2006), and 41 g/L (Parekh et al. 1987)

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in pretreated aspen wood. However, P. stipitis is not a very potent ethanol producer as its maximum ethanol productivity is only around 0.9 g/L/h (Jeffries 1996). The ability of P. stipitis to consume acetic acid and reduce the furan ring in furfural and hydroxymethylfurfural (HMF) creates an opportunity for this yeast to clean up some of the most concentrated toxins in cellulosic biomass conversion (Agbogbo and Coward-Kelly 2008). This could be very beneficial in waste water treatment since there will be a reduction in the quantity of toxins to be treated. Recently, the genome sequence for P. stipitis was published (http://www.jgi.doe.gov/pichia) (Jeffries et al. 2007). The sequence showed numerous genes for bioconversion such as xylanase, endo-1,4-β-glucanase, exo-1, 3-β-glucosidase, β-mannosidase, and α-glucosidase. The presence of these genes in P. stipitis offers very useful traits for simultaneous saccharification and fermentation of cellulose and hemicellulose. The xylanolytic ability of P. stipitis was enhanced by the co-expression of xylanase and xylosidase-encoding genes (Den Haan and Van Zyl 2003). The resulting strains displayed improved biomass production on medium with birchwood glucuronoxylan as sole carbohydrate source. The C. thermocellum endoglucanase-encoding gene celD (GH family 9) was also functionally expressed in P. stipitis under transcriptional control of the yeast's XYL1 gene promoter (Piotek et al. 1998). As P. stipitis produces its own β-glucosidase, this recombinant strain should theoretically have the ability to utilize amorphous cellulose as sole carbohydrate source although this aspect was not tested. Due to its wide substrate range and ability to detoxify certain inhibitors present in pretreated lignocellulosic materials, interest will remain high in enhancing the cellulolytic and hemicellulolytic abilities of this organism. Hansenula polymorpha Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) are able to ferment glucose, cellobiose, and xylose to ethanol (Ryabova et al. 2003). Furthermore, H. polymorpha is likely the only known thermotolerant methylotrophic yeast with an optimal growth temperature of 37 °C and maximal growth temperatures up to 48 °C. During the last decades, this yeast has been a favorable model organism to study the genetic control mechanism of methanol metabolism and peroxisome biogenesis (Piskur et al. 2006). In addition, it has also become one of the most powerful microbial hosts for the production of commercially important recombinant proteins on an industrial scale (Romanos et al. 1992). The inducible methanol oxidase (MOX) promoter allows protein production of up to 37% of total cellular protein. Additionally, physiological characteristics of H. polymorpha, such as

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resistance to heavy metals and oxidative stress, make this yeast attractive for several biotechnological purposes. These attributes, wide substrate range for soluble sugars present in lignocellulosic biomass, thermotolerance, process hardiness and a high capacity for heterologous protein production make this yeast an attractive candidate for CBP organism development. H. polymorpha was shown to produce high volumetric levels of endoglucanases from genes originating from the fungi A. aculeatus and Humicola insolens (Müller et al. 1998). A thermostable endoglucanase was also successfully produced in this yeast (Papendieck et al. 2002). As H. polymorpha is capable of growth on cellobiose, these recombinant strains should theoretically have the ability to hydrolyse amorphous cellulose although this aspect was not tested. A recent report further highlighted the promise of H. polymorpha in biomass conversion when strains were constructed that could ferment starch and xylan (Voronovsky et al. 2009). The T. reesei xyn11B gene encoding an endoxylanase was co-expressed in H. polymorpha with Aspergillus niger xlnD (GH family 3) coding for β-xylosidase under control of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 °C, demonstrating the promise of this organism in biomass conversion.

Engineering prokaryotic organisms to hydrolyze polysaccharides Escherichia coli Escherichia coli is a very well-studied organism and is therefore an ideal starting point for genetic engineering of a microorganism capable of producing bio-ethanol from cellulose. Although E. coli cannot hydrolyze cellulose or produce ethanol at appreciable quantities, it has been shown to metabolize all major sugar monomers present in plant biomass (Alterthum and Ingram 1989). During glycolysis, cells produce ATP and NADH by converting glucose and other simple sugars to pyruvate (Ingram et al. 1987). Pyruvate is in turn consumed to regenerate NAD+ to sustain glycolysis for further ATP production. The end products of these pathways are often referred to as fermentation products and vary greatly among different microorganisms. Products include organic acids like lactate, acetate, succinate, butyrate, and neutral products like ethanol, butanol, acetone, and butanediol. In wild-type strains of E. coli pyruvate is converted to acetyl-CoA and formate (Ingram et al. 1997). Acetyl-CoA is subsequently reduced to acetaldehyde by an aldehyde dehydrogenase and then to ethanol by an alcohol dehydro-

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genase. Both these steps require NADH and the conversion of an equal amount of acetyl-CoA to acetate to maintain redox balance. Therefore, in native E. coli, only half of the available pyruvate is converted to ethanol. Ethanolproducing organisms like S. cerevisiae and Zymomonas mobilis convert pyruvate directly to acetaldehyde by means of a pyruvate decarboxylase, allowing them to produce one mole of ethanol for every mole of pyruvate. Bräu and Sahm (1986) successfully expressed the Z. mobilis pyruvate decarboxylase at high levels in E. coli. The level of ethanol produced by this strain was comparable with that obtained in Z. mobilis. Subsequent work has focused on improving ethanol yields (Chen et al. 2009; Da Silva et al. 2005; Ingram et al. 1987, 1991; Ohta et al. 1991a), growth rates (Ingram et al. 1987), strain stability (Ohta et al. 1991a), and ethanol tolerance (Yamano et al. 1998) as well as increasing the range of substrates E. coli can ferment (Alterthum and Ingram 1989; Bräu and Sahm 1986; Burchhardt and Ingram 1992; Da Silva et al. 2005; Wood et al. 1997). These recombinant E. coli strains (E. coli KO11) require simple fermentation conditions; they produce higher concentrations of ethanol and are more efficient than pentose-fermenting yeast (Table 1). Some E. coli strains do have cryptic genes for cellobiose metabolism; however, none of these are capable of rapid growth on cellobiose (Moniruzzaman et al. 1997). Klebsiella oxytoca contains a phosphoenol-dependent phosphotransferase system (PTS) enabling it to utilize cellobiose. The K. oxytoca casAB operon coding for an enzyme IIcellobiose and a phospho-β-glucosidase was expressed in the ethanol-producing strain of E. coli (KO11). Unfortunately, expression was very poor. However, spontaneous mutants were isolated, which exhibited more than 15 times higher specific activities for cellobiose metabolism. The best mutant produced 45 g/L ethanol with a yield of 94% of the theoretical maximum. Two of these mutants were tested for their ability to produce ethanol from mixed waste office paper in a simultaneous saccharification and fermentation (SSF) process. Here the best mutant produced 32.7 g/L ethanol with a yield of 72% of the theoretical maximum. Cellulases have recently been engineered into these ethanol-producing strains, thereby reducing the amount of

externally added enzymes needed to convert plant cellulose in a SSF process to ethanol. Numerous endoglucanases have been expressed in E. coli allowing it to hydrolyze amorphous and soluble cellulose to shorter cellooligosaccharides (Da Silva et al. 2005; Seon Park et al. 2007; Srivastava et al. 1995; Wood et al. 1997; Yoo et al. 2004; Zhou et al. 1999). Among the enzymes successfully produced are Cel5Z and Cel8Y from Erwinia chrysanthemi (Zhou et al. 1999; Zhou and Ingram 2000). In E. chrysanthemi, Cel5Z accounts for more than 95% of the activity on CMC and Cel8Y attributes 5%. Unfortunately E. coli has limited ability to secrete proteins into the extracellular medium and as a result most of the recombinant Cel5Z accumulated in the periplasmic space, while 90% of Cel8Y was secreted as an extracellular product (Wood et al. 1997). At least three different types of protein secretion systems have been identified in Gram-negative bacteria. Of these, the most widely used for protein secretion is type II. It employs a two-step process in which the N-terminal signal peptide is cleaved from the pro-protein once it has been exported to the periplasmic space. The mature peptide is then secreted through the cell wall and outer membrane into the extracellular medium. Zhou et al. (1999) successfully reconstructed the type II secretion system, encoded by the out genes from E. chrysanthemi, in E. coli. This enabled E. coli to secrete more than 50% of the recombinant Cel5Z. Using purified Cel8Y and Cel5Z from the recombinant E. coli strain, Zhou and Ingram (2000) were able to show good synergy between these endoglucanases since Cel8Y and Cel5Z have very different substrate specificities. Cel8Y does not hydrolyze short cellooligosaccharides, but it is active on CMC, producing fragments averaging 10.7 glucosyl residues. Cel5Z is active on both short cello-oligosaccharides and amorphous cellulose, producing mainly cellobiose and cellotriose. Together, Cel8Y and Cel5Z show good synergism, and CMC was hydrolyzed to products averaging 2.3 glucosyl residues. E. coli strains capable of breaking down cellulose could potentially also be genetically modified to produce other commodity products like lactic acid (Chang et al. 1999; Zhou et al. 2003), succinic acid (Lin et al. 2005; Sanchez et al. 2005), or acetic acid (Causey et al. 2002) (Table 2).

Table 1 Ethanol production by recombinant E. coli strains

Parameter

Sugar

Amount

Reference

Yield (g/g sugar)

Glucose Xylose Glucose Xylose Glucose Xylose

0.57 0.47 2.50 0.87 2.10 2.24 6%

(Da Silva et al. 2005; Ohta et al. 1991a) (Ohta et al. 1991a; Yamano et al. 1998) (Ohta et al. 1991a; Zaldivar et al. 2001) (Zaldivar et al. 2001) (Alterthum and Ingram 1989; Kim et al. 2007) (Kim et al. 2007) (Yamano et al. 1998)

Volumetric productivity (g/L/h) Specific productivity (g/gDCW/h) Ethanol tolerance

Appl Microbiol Biotechnol (2010) 87:1195–1208 Table 2 Commodity producing recombinant E. coli strains

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Commodity

Specific productivity (mmol/g/h)

Volumetric productivity (mmol/L/h)

Lactic acid Succinic acid Acetic acid

36

24.5

10

Klebsiella oxytoca is a hardy prototrophic bacterium with the ability to transport and metabolize cellobiose, cellotriose, xylobiose, xylotriose, sucrose, and all other monomeric sugars present in lignocellulosic biomass (Zhou and Ingram 1999). Four fermentation pathways are present in K. oxytoca: the pyruvate formate-lyase pathway that produces formate (hydrogen and carbon dioxide) and acetate plus ethanol in equimolar amounts, the lactic acid pathway, the succinate pathway, and the butanediol pathway (Ohta et al. 1991b). As with E. coli, it is possible to direct more than 90% of the carbon from sugar metabolism to ethanol. A recombinant strain, K. oxytoca P2, containing the Z. mobilis pdc and adhB genes was able to produce ethanol from soluble sugars at 95% of the maximum theoretical yield (Wood and Ingram 1992) (Table 3). Unlike most other ethanol-producing organisms, K. oxytoca has the ability to ferment xylose and glucose at equivalent rates (Ohta et al. 1991b). This significantly shortens the time required to ferment mixtures of glucose and xylose, typically present in lignocellulosic hydrolysates. In a comparative study, it was shown that K. oxytoca produced ethanol from cellobiose up to a concentration of 33 g/L, at a faster rate than Saccharomyces pastorianus, K. marxianus, and Z. mobilis (Golias et al. 2002). However, the maximum ethanol concentration measured was 37 g/L. This was slightly less than for other organisms that are more ethanol tolerant. A number of different cellulases have been expressed in ethanol-producing K. oxytoca strains (Moniruzzaman et al. 1997; Wood and Ingram 1992; Zhou and Ingram 1999, 2001; Zhou et al. 2001). Zhou and Ingram (1999, 2001) constructed a K. oxytoca strain expressing the E. chrysanthemi cel8Y and cel5Z endoglucanase genes. By also introducing the out genes that encodes the type II secretion system from E. chrysanthemi, both Cel8Y and

Reference

(Shanmugam and Ingram 2008) (Shanmugam and Ingram 2008) (Causey et al. 2002)

1.7

Klebsiella oxytoca

Table 3 Ethanol production by recombinant K. oxytoca strains

Yield (mol/mol glucose)

9.5

Cel5Z were secreted effectively by K. oxytoca. This strain produced more than 24,000 U/L of endoglucanase activity and was capable of fermenting amorphous cellulose and producing a small amount of ethanol without the addition of cellulases. With the demand for alternative fuels, ethanol has become an important commodity; however, K. oxytoca has also been engineered to produce 2, 3-butanediol (Ji et al. 2009). Zymomonas mobilis Zymomonas mobilis is a well known ethanol-producing bacterium which has been used historically in tropical areas to make alcoholic beverages from plant sap (Yanase et al. 2007). Like other ethanol-producing bacteria it has attracted much attention in recent years because of the high rate at which it produces ethanol. In addition to this it exhibits many other traits required for CBP. It has a high tolerance for ethanol, is able to ferment sugars at low pH and has good resistance to the inhibitors found in lignocellulosic hydrolysates. Unfortunately, Z. mobilis has two major shortcomings in that it does not ferment or utilize xylose as carbon source and it cannot hydrolyze polysaccharides. It is however relatively easy to genetically manipulate. Zhang et al. (1997) patented a Z. mobilis strain capable of fermenting both xylose and arabinose, the major pentose sugars present in plant material. This required the introduction of seven different genes encoding xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphate 4-epimerase, transaldolase, and transketolase. Co-fermentation of 100 g/L sugar (glucose:xylose:arabinose—40:40:20) yielded a final ethanol concentration of 42 g/L in 48 h. Although co-fermentation was achieved, there was a preferential order of utilization: glucose first, then xylose and lastly arabinose (Mohagheghi et al. 2002).

Parameter

Sugar

Amount

Reference

Yield (g/g sugar)

Glucose Xylose Glucose Xylose

0.52 0.48 2.10 2.00 3.7%

(Ohta et al. 1991b) (Ohta et al. 1991b) (Ohta et al. 1991b) (Ohta et al. 1991b) (Golias et al. 2002)

Volumetric productivity (g/L/h) Ethanol tolerance

1202

A number of papers have been published on the production of ethanol from cellulosic biomass with Z. mobilis using simultaneous saccharification and fermentation (Eklund and Zacchi 1995; Gutierrrez-Padilla and Karim 2005; Kademi and Baratti 1996; Park et al. 1993; Yamada et al. 2002); however, few papers have been published on recombinant Z. mobilis strains capable of breaking down cellulose and fermenting it to ethanol. Brestic-Goachet et al. (1989) expressed Erwinia chrysanthemi cel5Z in Z. mobilis. The maximum activity obtained was 1,000 IU/L with 89% of the recombinant endoglucanase secreted to the extracellular medium. This is significantly more than the results obtained with the Pseudomonas fluorescens eglX endoglucanase (Brestic-Goachet et al. 1989; Lejeune et al. 1988). Expression of the Ruminococcus albus β-glucosidase enabled Z. mobilis to ferment 20 g/L cellobiose to 10.7 g/L ethanol in 2 days (Yanase et al. 2005). Clostridium acetobutylicum Strains of C. acetobutylicum have been used for the large scale production of acetone and butanol via the acetone/ butanol/ethanol (ABE) pathway (Jones and Woods 1986). With the renewed interest in biofuels, C. acetobutylicum strains are again under the magnifying glass for the costeffective production of butanol. However, one drawback of C. acetobutylicum strains is their inability to grow on crystalline cellulose (Perret et al. 2004). Nevertheless, some C. acetobutylicum strains produce endoglucanase and cellobiase activity and can grow on amorphous cellulose and cellobiose (Lee et al. 1985; Allcock and Woods 1981; Lopez-Contreras et al. 2004). Sequence analysis of the genome of C. acetobutylicum ATCC 824 showed a cellulosome-like gene cluster containing ten transcribed genes that are predicted to encode secreted proteins with cohesin and dockerin modules and that it is capable of producing small amounts of a 665-kDa cellulosome devoid of activity towards crystalline cellulose and with poor activity against CMC or PASC (Nölling et al. 2001). Further analysis demonstrated at least four components, the scaffoldin CipA, and four cellobiohydrolases Cel48A, Cel9X, and Cel9C/Cel9E (Sabathe and Soucaille 2003) are produced. The CipA was demonstrated to be functional, and C. acetobutylicum is therefore suited for the production, secretion, and assembly of heterologous mini-cellulosomes (Mingardon et al. 2005; Sabathe et al. 2002). Lopez-Contreras et al. (2003, 2004) found that C. acetobutylicum ATCC 824 produced higher extracellular levels of cellulases Cel9G and in particular cellobiohydrolase Cel48F, which was identified as a major component in other Clostridium cellulosomes, when grown on lichenan or xylose, but not on glucose or cellobiose.

Appl Microbiol Biotechnol (2010) 87:1195–1208

Different groups are currently trying to express recombinant cellulases from C. cellulolyticum (e.g., Cel48F and Cel9G) to be assembled on chimeric mini-scaffoldin constructs in C. acetobutylicum in an attempt to generate recombinant strains capable of utilizing crystalline cellulose (Perret et al. 2004). Lactic acid bacteria Lactic acid bacteria are common inhabitants in animal digestion tracts and participate in grass silage by converting excess sugars to lactic acid and prevent further deterioration (Moon 1984; Weinberg and Muck 1996). Addition of cellulases to grass silage or to monogastric animal (such as poultry and pigs) feeds improved conversion and digestibility. The expression of cellulases in non-cellulolytic lactic acid bacteria could potentially contribute to grass silage or improve digestibility of non-soluble polysaccharides in feeds (Ozkose et al. 2009). Different groups expressed bacterial cellulases in Lactobacillus plantarum, often used as primary inoculants for silage fermentations. The endo-1,4-β-glucanase genes of C. thermocellum (cel5C) and a Bacillus sp. (celA) were successfully expressed in L. plantarum, but low cellulolytic activities were measured (Bates et al. 1989; Rossi et al. 2001; Scheirlinck et al. 1989; Sharp et al. 1992). It is noteworthy that recombinant L. plantarum expressing these cellulases still dominated silage fermentations and Rossi et al. (2001) recorded increased acidification capacity that could improve silaging. Different cellulases have also been introduced in Lactobacillus species associated with the digestion tract. The endo-1,4-β-glucanase gene of Piromyces rhizinflata (eglA, GH family 5) and endo-1,3-1,4-β-glucanases genes of Bacillus macerans (bglM) and Fibrobacter succinogenes were expressed in Lactobacillus reuteri (Heng et al. 1997; Liu et al. 2005, 2007), as well as the endo-1,4-β-glucanase gene of C. thermocellum (cel5C) in Lactobacillus gasseri, Lactobacillus johnsonii (Cho et al. 2000) and the 1,3-1,4-βglucanase gene of Bacillus amyloliquefaciens (bglA, GH family 1) in Lactobacillus crispatus, Lactobacillus brevis, and Lactobacillus fermentum (Sieo et al. 2005). The use of the recombinant L. crispatus, L. brevis, and L. fermentum in broiler chicken feed experiments showed improved feed passage rates. Thus, although the expression of individual cellulases in lactic acid bacteria did not render them cellulolytic, their performance in grass silage and to facilitate better digestion was demonstrated. Corynebacteria Corynebacteria have traditionally been used for industrial amino acid production. The endoglucanase (celA1 GH family 6) gene of Streptomyces hastedii JM8 and the

Appl Microbiol Biotechnol (2010) 87:1195–1208

exoglucanase (cex, GH family 10) and endoglucanase (cenA GH family 6) genes of Cellulomonas fimi have been successfully expressed in Brevibacterium lactofermentum (Adham et al. 2001; Paradis et al. 1987). The expression of cellulases in this important group of bacteria could potentially unlock the use of abundant cellulosic materials for amino acid production in the future.

Discussion Strain development is the most important technical obstacle towards the conversion of lignocellulose to commodity products in a CBP configuration (Alfenore et al. 2002; Bothast et al. 1999). Organisms with broad substrate ranges and cellulolytic and/or hemicellulolytic abilities generally suffer from poor growth characteristics or poor productproducing characteristics. These include poor yield, titer, and rate or producing mixtures of products where desirable products are produced along with undesirables. In comparison, organisms with desirable product-producing qualities often suffer from limited substrate range including lack of cellulolytic ability, poor fermentation qualities, and sensitivity to the inhibitors present in pretreated lignocellulosic biomass. The ideal CBP organism should be robust, able to degrade lignocellulose, and utilize hexose and pentose sugars at high efficiency. To date, no such organism has been identified. Bacteria generally have a high growth rate but lack process robustness. Yeasts are often sufficiently robust but lack substrate range. Filamentous fungi often have a wide substrate range but grow relatively slowly and do not produce enough of a desirable product. The engineered organisms discussed in this review are compared in Table 4 with regard to improved performance. For ethanol production, the main shortcoming of traditional microorganisms used for fermentation such as the yeast S. cerevisiae and the bacterium Z. mobilis is their inability to metabolize pentose sugars or sugar polymers. Relatively efficient xylose-metabolizing strains for both these organisms have been constructed over the past 20 years. Furthermore, considerable attention has also been given to the natural xylose-fermenting yeast P. stipitis, but this organism is not an efficient ethanol producer (Hahn-Hagerdal et al. 2007; Jeffries et al. 1996). However, P. stipitis does have the ability to detoxify some of the inhibitors found in most lignocellulose hydrolysates (Agbogbo and Coward-Kelly 2008). The yeast K. marxianus exhibits many of the favorable characteristics of S. cerevisiae, with the added advantage of being able to ferment xylose directly to ethanol under anaerobic conditions (Margaritis and Bajpai 1982). Furthermore, K. marxianus has the ability to grow at temperatures of up to 52 °C, allowing for more efficient

1203

enzymatic hydrolysis and reducing both the risk of contamination and the need to cool the pretreated material before fermentation (Pecota et al. 2007). Many strains of this organism can grow on a variety of inexpensive carbon sources, making them economically attractive for commercial processes. H. polymorpha can also grow at elevated temperatures and has the ability to produce vast amounts of heterologous protein, which could be advantageous in creating a cellulolytic system in this organism. While the advantages of using the yeasts S. cerevisiae, P. stipitis, K. marxianus, and H. polymorpha are well appreciated, the engineered cellulolytic ability of all of these strains is at best rudimentary. None of the strains is as yet capable of utilizing crystalline cellulose, and the high level production of an exocellulase remains a requirement. New information on secretion pathways, chaperones, and metabolic engineering should help alleviate this problem in future. Numerous E. coli and K. oxytoca strains have been constructed that efficiently ferment pentose and hexose sugars to ethanol (Alterthum and Ingram 1989; Burchhardt and Ingram 1992; Hahn-Hagerdal et al. 2007; Kim et al. 2007; Ohta et al. 1991a, b; Yamano et al. 1998; Zhou and Ingram 1999, 2001). Among all the abovementioned organisms, the recombinant bacteria are the most productive. However, compared to S. cerevisiae, all of the bacterial species discussed in this review are relatively sensitive to inhibitors associated with lignocellulosic hydrolysates (Bothast et al. 1999; Ohta et al. 1991b; Yamano et al. 1998). Engineering-enhanced protein secretion in E. coli allowed the successful secretion of endoglucanases (Zhou et al. 1999). Zhou and Ingram (1999, 2001) also constructed a K. oxytoca strain effectively secreting endoglucanases, and this strain was capable of fermenting amorphous cellulose and producing a small amount of ethanol. E. coli and K. oxytoca strains capable of breaking down cellulose could also be modified to produce other commodity products like lactic acid (Chang et al. 1999; Zhou et al. 2003), succinic acid (Lin et al. 2005; Sanchez et al. 2005), acetic acid (Causey et al. 2002), or 2,3-butanediol (Ji et al. 2009). Further development of C. acetobutylicum, lactic acid bacteria, and Corynebacteria species for cellulose utilization could unlock the potential of using biomass as a feedstock for commodity product production. There are also other organisms under development for biomass conversion. Among these is the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum that grows in a temperature range of 45–65 °C and a pH range of 4.0–6.5 (Shaw et al. 2008a). It is able to ferment a wide range of sugars present in cellulosic biomass, including cellobiose, glucose, xylose, mannose, galactose, and arabinose. Unlike most organisms, T. saccharolyticum metabolizes xylose and glucose essentially at the same rate (Shaw et

+++ +++ +++ ++

+

+++

+++

−

Resistant to hydrolysate inhibitors GRAS status

Low pH

High temperature

NA Not available

E+

−

++

Ferment xylose

+++

+++

Grow on glucose E+++

+++

−

−

+++

−

Utilize xylobiose

E+++

++

−

Grow on xylose

+++

−

Ferment glucose

+

−

Breakdown crystalline cellulose Breakdown amorphous cellulose Breakdown hemicellulose Utilize cellobiose

+++

+++

+++

++

E+

E++

+++

+++

+++

−

−

−

−

WT

GE

WT

+++

+++

+++

++

E+

E++

+++

+++

+++

+++

+

+

−

GE

K. marxianus

+++ ++ + −

S. cerevisiae

Good activity Low activity Very low activity No activity

−

+++

+++

+

E+++

E+

+++

+++

+++

+++

+

−

−

WT

P. stipitis

−

+++

+++

+

E+++

E+

+++

+++

+++

+++

++

++

−

GE

+++

+++

+++

+

E++

E++

+++

+++

−

+++

−

−

−

WT

+++

+++

+++

+

E++

E++

+++

+++

+++

+++

++

++

−

GE

H. polymorpha

−

− −

−

+

++

−

+

E+

E+++

−

+++

−

+++

+

+

+

+

−

GE

+++

+++

−

−

−

−

−

WT

E. coli

−

+

−

−

−

−

+++

+++

+++

+++

−

−

−

WT

−

+

−

+

E+++

E+++

+++

+++

+++

+++

+

+

−

GE

K. oxytoca

−

+

+++

+

−

E+++

−

+++

−

−

−

−

−

WT

−

+

+++

+

E+

E+++

+

+++

+

+

NA

++

−

GE

Z. mobilis

++

+++

NA

NA

B+

B+

+++

+++

+++

+++

+

++

−

WT

++

+++

NA

NA

B+

B+

+++

+++

+++

+++

+

+

−

GE

C. acetobutylicum

++

+++

+++

NA

L++

L++

+++

+++

NA

+++

++

++

−

WT

++

+++

+++

NA

L++

L++

+++

+++

NA

+++

++

+

−

GE

Lactic acid bacteria

+++

NA

NA

NA

−

−

+++

+++

+++

+++

−

−

−

WT

+++

NA

NA

NA

E+++

E+++

+++

+++

+++

+++

−

−

−

GE

T. saccharolyticum

Table 4 Comparison of several candidate CBP organisms. For each organism the wild type (WT) and the genetically engineered (GE) strain properties are compared. The main fermentation products on glucose and xylose are indicated as E (ethanol), B (butanol) or L (lactic acid)

1204 Appl Microbiol Biotechnol (2010) 87:1195–1208

Appl Microbiol Biotechnol (2010) 87:1195–1208

al. 2008a, b). Like all thermophilic saccharolytic anaerobes described to date, it produces organic acids in addition to ethanol. Knockout mutants were obtained for T. saccharolyticum that produced almost exclusively ethanol from xylose. T. saccharolyticum naturally produces both a xylanase (Lee et al. 1993a, b) and a β-xylosidase (Yang et al. 2004) enabling it to ferment xylan directly to ethanol. Using Avicel as carbon source, T. saccharolyticum was able to produce as much ethanol from Avicel with four filter paper units (FPU) of externally added enzyme as S. cerevisiae was with ten FPU in SSF, the result of improved enzyme efficiency at higher temperatures (Shaw et al. 2008b). This shows the potential of this thermophile as CBP organism if a cellulolytic system can be established. While there is still no ideal organism to use in a CBP configuration for biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. It is likely that more than one organism may eventually be used in various biomass conversion processes, and the choice may depend on the sugar composition of the feedstock, the pretreatment method used and the end product required.

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