Scholarly article on topic 'Engineering Sugar Utilization and Microbial Tolerance toward Lignocellulose Conversion'

Engineering Sugar Utilization and Microbial Tolerance toward Lignocellulose Conversion Academic research paper on "Biological sciences"

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Academic research paper on topic "Engineering Sugar Utilization and Microbial Tolerance toward Lignocellulose Conversion"



published: 18 February 2015 doi: 10.3389/fbioe.2015.00017

Engineering sugar utilization and microbial tolerance toward lignocellulose conversion

Production of fuels and chemicals through a fermentation-based manufacturing process that uses renewable feedstock such as lignocellulosic biomass is a desirable alternative to petrochemicals. Although it is still in its infancy, synthetic biology offers great potential to overcome the challenges associated with lignocellulose conversion. In this review, we will summarize the identification and optimization of synthetic biological parts used to enhance the utilization of lignocellulose-derived sugars and to increase the biocatalyst tolerance for lignocellulose-derived fermentation inhibitors. We will also discuss the ongoing efforts and future applications of synthetic integrated biological systems used to improve lignocellulose conversion.

Keywords: synthetic biology, metabolic engineering, lignocellulose, xylose, furan aldehydes

Lizbeth M. Nievesf, Larry A. Panyonf andXuan Wang*

School of Life Sciences, Arizona State University, Tempe, AZ, USA Edited by:

Pablo Carbonell, University of Evry, France

Reviewed by:

Weiwen Zhang, Tianjin University, China

Taek Soon Lee, Lawrence Berkeley National Laboratory, USA


Xuan Wang, School of Life Sciences, Arizona State University, 427 E. Tyler Mall, Tempe, AZ 85287, USA e-mail:

* Lizbeth M. Nieves and Larry A. Panyon have contributed equally to this work.


One of the daunting challenges faced by the modern world is our unsustainable dependence on petroleum as the primary source for transportation fuels and many chemical products including solvents, fertilizers, pesticides, and plastics (Service, 2007). To fulfill future societal needs, we have to find a sustainable supply of energy and chemicals. Synthetic biology has emerged as a young discipline with the great potential to construct a novel biological system to produce fuels and chemicals from renewable sources in a cost-effective manner, thus ultimately achieving energy self-sufficiency independent of petroleum. We will apply the synthetic biology definition of "the design and construction of new biological components, such as enzymes, genetic circuits, and cells, or the redesign of existing biological systems" throughout this review (Keasling, 2008). The engineered biological systems created by synthetic biology include enzymes with new functions, genetic circuits, and engineered cells with unique specifications (Cameron et al., 2014; Way et al., 2014). In many cases, the ultimate goal is to rationally manipulate organisms to facilitate novel functions, which do not exist in nature (Cameron et al., 2014; Way et al., 2014). Thus far, synthetic biology has contributed to many fields such as bio-based production (Keasling, 2008; Jarboe et al., 2010), tissue and plant engineering (Bacchus et al., 2012; Moses et al., 2013; Xu et al., 2013; Trantidou et al., 2014), and cell-free synthesis (Lee and Kim, 2013).

Plant biomass (lignocellulose) represents arguably the most important renewable feedstock on the planet. Lignocellulose is a complex matrix of various polysaccharides, phenolic polymers, and proteins that are present in the cell walls of woody plants (Saha, 2003; Girio et al., 2010). Conversion of non-food plant biomass, especially agricultural residues such as corn stover and sugarcane bagasse, avoids the many concerns about the production of fuels and chemicals derived from food sources (Lynd, 1990).

Additionally, non-food-based biofuels offer greater cost reduction in the longer term (Lynd, 1990). For numerous types of agricultural residues, the sugar content is comparable to corn (Saha, 2003). However, the conversion of these sugars from agricultural residues to fuels and chemicals in a cost-effective manner still remains challenging. There are at least three major challenges to be solved before lignocellulose bioconversion becomes financially feasible (Figure 1). First, in contrast to starch, which is easily degraded into fermentable sugar monomers, sugars in lignocel-lulose are locked into very stable polymeric structures including cellulose and hemicellulose (Saha, 2003; Girio et al., 2010). These polymers are designed by nature to resist deconstruction (Alvira et al., 2010). The crystalline-like fibers of cellulose are encased in a covalently linked mesh of lignin and hemicellulose. Cellulose (3040% of biomass dry weight) is composed of only D-glucose linked by P-1,4 glycosidic bonds while a mixture of pentoses, especially D-xylose, and hexoses comprises the main component of hemicellulose (20-40% of biomass dry weight) (Saha, 2003). Lignin is not the saccharides polymer but a complex polymer of aromatic alcohols. Different types of lignocellulosic biomass vary in the composition of cellulose, hemicellulose, and lignin (Saha, 2003). Chemical pretreatment processes are commonly required for lignocellulose conversion. Steam pretreatment with dilute mineral acids is an efficient approach to depolymerize hemicellulose into sugar monomers and to increase the accessibility of cellu-lase enzymes to degrade cellulose (Saha, 2003; Sousa et al., 2009; Alvira et al., 2010). After pretreatment and cellulase digestion, most of the sugars in agricultural waste will be released into the broth and thus ready to be converted into fuels and chemicals if a suitable biocatalyst is applied. The cost of cellulase enzymes is currently still prohibitive to wide application of lignocellulose conversion. Continuing efforts of synthetic biologists from academic and industrial labs are improving cellulase enzymes or

Major Challenges

• Degradation recalcitrance

• Efficient xylose metabolism

• Fermentation inhibitors

conversion into fuels and chemicals at low cost (Sheridan, 2013). Until now, most efforts for lignocellulose conversion have been devoted to microbial ethanol production. By pathway engineering and metabolic engineering, the microbial hosts can extend their metabolism to produce valuable chemicals other than ethanol from lignocellulose. This review focuses on engineering new biological components by synthetic biology to improve lignocellulose conversion. The past efforts, current status, and future challenges will be discussed.

FIGURE 1 | Challenges of lignocellulose conversion. Lignocellulose regularly needs pretreatment to release its sugar components for biocatalysts to make fuels and chemicals. This is a sustainable approach to reduce our dependence on petroleum and to prevent carbon dioxide emission. At least three major challenges remain to be solved for a cost-effective lignocellulose conversion.

enzyme complexes aiming to develop catalysts that are cost- Synthetic biology has the potential to re-design microbial biology

effective enough to be suitable for commercialization. The recent to simultaneously use D-glucose and other pentoses efficiently.

advancements in cellulases have been extensively reviewed (Elkins Lignocellulosic raw materials commonly contain much higher

et al., 2010; Garvey et al., 2013; Hasunuma et al., 2013; Bommar- amounts of D-xylose compared to other pentoses, and therefore,

ius et al., 2014) and therefore are not the scope of this review. improving xylose fermentation has become a priority (Girioetal.,

Second, one of the major carbohydrates in the typical lignocellu- 2010). Xylose degradation is not universal for all microbes in spite

losic biomass is D-xylose, a five-carbon aldose, which is difficult of being the most abundant monosaccharide in hemicellulose. At

for many microbes to metabolize. For instance, common ethanol- the current stage, most related research still uses the trial-and-error

producing industrial microbes such as Saccharomyces cerevisiae approach to accelerate xylose transport and xylose metabolism.

and Zymomonas mobilis, do not natively metabolize xylose (Saha, A more quantitative understanding of sugar catabolism is nec-

2003). Although some microbes such as Escherichia coli and Kleb- essary before synthetic biologists are able to predict and design

siella pneumonia have the native xylose metabolic pathway, it is a biological system that efficiently transports and metabolizes

not efficient and is commonly repressed by the presence of glu- sugars.

cose (Saha, 2003). Third, side products that hinder cell growth and There are two major metabolic pathways to catabolize xylose: fermentation such as furfural, 5-hydroxymethylfurfural, formate, xylose isomerase pathway and oxidoreductase pathway used by acetate, and soluble lignin products are formed during common bacteria and fungi, respectively (Figure 2). These pathways have chemical pretreatment processes (Saha, 2003; Mills et al., 2009). been constructed and optimized in industrial biocatalysts such For example, furfural (dehydration product of pentose sugars) as S. cerevisiae and Z. mobilis, which cannot natively metabolize is widely regarded as one of the most potent inhibitors (Mills xylose. There are comprehensive reviews that excellently summa-et al., 2009; Geddes et al., 2010a, 2011). It can completely inhibit rized this research topic (Jeffries and Jin, 2004; Chu and Lee, 2007; cellular growth at low concentrations (Zaldivar et al., 1999; Liu Matsushika et al., 2009; Young et al., 2010; Cai et al., 2012; Kim and Blaschek, 2010). The concentration of furfural is correlated et al., 2013). Here, we only briefly review some of important past with the toxicity of dilute acid hydrolyzates (Martinez et al., efforts. The xylose oxidoreductase pathway is commonly used 2000). Overliming to pH 10 with Ca(OH)2 or active carbon fil- by some ascomycetous yeasts such as Pichia stipitis (Figure 2). ter reduces the level of furfural and toxicity, but increases the Although the S. cerevisiae chromosome has genes encoding xylose process complexity and operational cost, thus reducing economic reductase, xylitol dehydrogenase, and xylulokinase, their native viability (Martinez et al., 2000). There has been a growing inter- expression level is too low to support cellular growth when using est to engineer industrially related strains to be more resistant to xylose as the sole carbon source (Yang and Jeffries, 1997; Richard these inhibitors (Wang et al., 2012a,b; Zheng et al., 2012; Ged- et al., 2000; Traff et al., 2002; Toivari et al., 2004). Anaerobic xylose des et al., 2014; Xiao and Zhao, 2014). For example, beneficial fermentation by S. cerevisiae was first demonstrated by heterolo-genetic traits to increase host tolerance of furan aldehydes have gous expression of XYL1 (Rizzi et al., 1988) and XYL2 (Rizzi et al., been identified (Taherzadeh et al., 2000; Liu et al., 2004, 2005, 1989) genes encoding xylose reductase and xylitol dehydrogenase 2008; Gorsich et al., 2006; Petersson et al., 2006; Almeida et al., from P. stipitis (Kotter et al., 1990; Tantirungkij et al., 1994). How-2008; Geddes et al., 2014; Glebes et al., 2014a,b; Luhe et al., 2014), ever, the xylitol is accumulated as a significant side product when knowledge about toxicity mechanisms has been accumulated (Lin genes XYL1 and XYL2 are overexpressed in the recombinant S. et al., 2009a; Miller et al., 2009a,b; Ma and Liu, 2010; Glebes et al., cerevisiae, which lowers the ethanol yield. The accumulation of 2014a,b), and thus the integrated synthetic detoxification systems xylitol is likely due to the cofactor imbalance of the first two steps have been constructed and proven effective in different biocatalysts in the oxidoreductase pathway (Figure 2). NADPH is the preferred (Wang et al., 2013). cofactor for xylose reductase to reduce xylose, while NAD is used Despite government incentives and mandates, these grand chal- by xylitol dehydrogenase to oxidize xylitol, resulting in the forlenges have prohibited the commercialization of lignocellulose mation of xylulose (Figure 2). Unlike many bacteria, S. cerevisiae

FIGURE 2 |Two metabolic pathways of D-xylose metabolism. Xylose is transported into cells and then it is either isomerized by xylose isomerase in some bacteria or reduced to xylitol by xylose reductase in some fungi. Xylitol is oxidized to xylulose and then phosphorylated to form xylulose-5-phosphate by xylulokinase. Xylulose-5-phosphate enters the pentose phosphate pathway for further degradation. The isomerase pathway avoids the production of xylitol.

lacks pyridine nucleotide transhydrogenases, which catalyze the conversion between these two reducing cofactors, NADPH and NADH (Nissen et al., 2001). Therefore, this imbalance of cofac-tors caused by these two reactions will eventually lead to slow kinetics for xylose degradation and xylitol accumulation. Although overexpression of the xylose reductase and xylitol dehydrogenase genes has been shown to enable xylose metabolism in recombinant S. cerevisiae strains, overexpression of the xylulokinase gene is often required to create a complete functional heterologous pathway and to further reduce xylitol production (Ho et al., 1998; Jin et al., 2005; Bettiga et al., 2008). One successful example of engineering an efficient xylose-metabolizing yeast is the recombinant Saccharomyces sp. strain 1400(pLNH32) (Ho et al., 1998). In this strain, the P. stipitis xylose reductase, P. stipitis xylitol dehydrogenase, and S. cerevisiae xylulokinase genes under the control of the strong native glycolytic promoters were cloned into the plasmid pLNH32 to achieve high expression level. The aerobic conversion of xylose to ethanol has relatively high titer (23 g/L), yield (~0.45 g ethanol/g xylose, theoretic yield is ~0.5 g ethanol/g xylose for ethanol fermentation), and productivity (4g/L/h) in a complex medium (Ho et al., 1998). Further improvements of ethanol titer and yields in several xylose-fermenting industrial yeast strains such as TMB 3400 and 424A(LNF-ST) have been achieved by utilizing the heterologous xylose oxidoreductase pathway and other genetic modifications that enhance the downstream pentose phosphate pathway (Matsushika et al., 2009). This demonstrates the potential of the xylose oxidoreductase pathway to improve xylose metabolism.

The xylose isomerase pathway, dominantly used by many bacteria including E. coli and Bacillus subtilis, has also been constructed in S. cerevisiae strains. In this pathway, xylose is directly converted to xylulose through a one-step reaction catalyzed by xylose isomerase or other aldose isomerases (Figure 2). This pathway does

not involve xylitol formation and it does not require a reducing cofactor. However, this isomerization reaction thermodynamically favors xylose over xylulose at equilibrium (Jeffries, 1983), which requires an alternative driving force such as efficient downstream reactions to promote the equilibrium moving toward the formation of xylulose (Figure 2). In addition, it has been shown that the expression of functional bacterial xylose isomerase genes often result in inefficient enzymatic activities and thus low xylose utilization (Sarthy et al., 1987; Gardonyi and Hahn-Hagerdal, 2003). The unsuccessful heterologous expression is probably due to the protein misfolding and post-transcriptional modification. Even though the successful synthesis of active xylose isomerases derived from different microbes including thermophilic bacterium Thermus thermophiles (Walfridsson et al., 1996), Piromyces sp.E2 (Kuyper et al., 2003), Orpinomyces (Madhavan et al., 2009), and Clostridium phytofermentans (Brat et al., 2009) has been achieved in S. cerevisiae at high levels, the rate of growth on xylose was still poor. It is possible that further optimization is needed to increase metabolic flux of downstream reactions, especially the pentose phosphate pathway. Ethanol yield is often higher in these recombinant S. cerevisiae using the xylose isomerase pathway than those using the heterologous xylose oxidoreductase pathway because xylitol production is avoided. However, the titer and productivity of S. cerevisiae using the xylose isomerase pathway are very low. The Piromyces sp. xylose isomerase has been extensively engineered to increase catalytic efficiency, and the S. cerevisiae BY4741-S1 derivatives expressing this mutant enzyme improved both its aerobic growth rate and ethanol production (Lee et al., 2012). However, in terms of xylose utilization and ethanol production, these optimized recombinant S. cerevisiae strains still perform more poorly with a final ethanol titer lower than 4 g/L. The heterologous xylose isomerase pathway has also been successfully constructed in other biocatalysts such as Z. mobilis, a bacterium notable for its bioethanol-producing capabilities, which has been used as a natural fermentative agent in alcoholic beverage production (Skotnicki et al., 1983). Similar to S. cerevisiae, Z. mobilis cannot metabolize xylose, which limits its application in lignocellulose conversion. In addition, Z. mobilis metabolizes glucose into pyruvate using the Entner-Doudoroff pathway instead of glycolysis (Embden-Meyerhof-Parnas pathway) and then converts pyruvate into ethanol and CO2 (Conway, 1992). Even with the successful expression of the xylose isomerase and xylulokinase genes from Xanthomonas campestris or Klebsiella pneumoniae, Z. mobilis was still unable to grow using xylose as the sole carbon source (Liu et al., 1988; Feldmann et al., 1992). Interestingly, in addition to overexpression of the xylose isomerase and xylulokinase genes, overexpression of the transal-dolase and transketolase genes (the main enzymes in the pentose phosphate pathway) resulted in a recombinant Z. mobilis with a functional xylose metabolism (Zhang et al., 1995). The resulting strain CP4 (pZB5) is able to convert xylose to ethanol with a higher titer (11 g/L) and yield (0.44 g/g xylose) compared to recombinant S. cerevisiae using the xylose isomerase pathway (Zhang et al., 1995). This excellent work strongly suggests a high flux of downstream metabolic reactions such as the pentose phosphate pathway is required for a functional xylose catabolism using the xylose isomerase pathway (Figure 2). A high performance

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of xylose to ethanol conversion using a bacterial xylose iso-merase pathway has been achieved in a wild-type E. coli strain (ATCC9637) after extensive metabolic engineering and adaptive laboratory evolution (Jarboe et al., 2007). The recombinant E. coli strain LY180 uses the native xylose isomerase pathway and the Z. mobilis ethanol-producing pathway to achieve the efficient conversion of xylose to ethanol with a high titer (45 g/L after 48 h) and yield (0.48 g/g xylose) using mineral salts medium (Miller et al., 2009b; Yomano et al., 2009). These successful examples of engineering Z. mobilis and E. coli suggest that the bacterial xylose isomerase pathway has the potential for efficient xylose conversion when the metabolic flux in downstream pathways is efficient.

Another challenge for the conversion of sugars derived from lignocellulose is the sequential metabolism of sugar mixtures, a phenomenon called catabolite repression. D-glucose represses the utilization of other sugars such as xylose in many industrial catalysts, thus impeding the rapid and complete utilization of sugar mixtures during fermentation. The mechanism of glucose repression is very complex and involves multiple levels of regulation. For example, E. coli has complex glucose repression mechanisms mainly through cyclic AMP, cyclic AMP-binding protein and enzymes of the phosphotransferase system (Kim et al., 2010). There are also other mechanisms involving the inhibition of transport of alternative sugars and a dual transcriptional regulator called Cra (Ramseier, 1996). Strains with the relaxed glucose repression should be able to simultaneously use a heterogeneous sugar mixture. However, genetic perturbation of glucose repression components can disrupt regular glucose metabolism and result in decreased glucose metabolism. It is challenging to engineer a biocatalyst with relaxed glucose repression while keeping a high glucose utilization rate. There are different engineering strategies developed to improve sugar co-utilization (Yomano et al., 2009; Chiang et al., 2013). In a recombinant E. coli strain, a com-binatory engineering strategy has achieved efficient co-utilization of glucose and xylose (30 g/L for each) in 16 h (Chiang etal., 2013). This genetic engineering strategy includes (1) deletion of ptsG (the glucose permease in phosphotransferase system) to release catabo-lite repression; (2) overexpression of a glucose transporter from Z. mobilis to restore glucose transport and metabolism; (3) overexpression of genes rpiA, tktA, rpe, and talB to increase pentose phosphate pathway. Recently, a completely different approach to decrease glucose repression has been developed (Galazka et al., 2010; Ha et al., 2011). Cellodextrins are glucose polymers of varying length (two or more glucose monomers) resulting from degradation of cellulose. Wild-type S. cerevisiae cannot assimilate cellodextrin because it lacks both the cellodextrin transporter and в-glucosidase capable of hydrolyzing cellodextrin into glucose. By integrating efficient transporters, the complemented hydrolytic enzymes for cellodextrin and the xylose oxidoreductase pathway (Figure 2) into S. cerevisiae, this recombinant S. cerevisiae strain is able to simultaneously consume cellodextrin and xylose probably because the glucose concentration is never high enough to induce the catabolite repression phenotype (Ha et al., 2011). It is plausible that intracellular hydrolysis of cellodextrin minimizes glucose repression of xylose fermentation allowing this co-consumption (Galazka et al., 2010; Ha et al., 2011). This novel strategy has

the potential to enable efficient co-utilization of sugar mixtures derived from lignocellulose.

Successful lignocellulose conversion requires efficient transport of the mixture of sugars into the cells. The transport of xylose is less efficient than the transport of glucose and often inhibited by D-glucose, which suggests xylose transport is a limiting factor for lignocellulose conversion (Jeffries and Jin, 2004; Luo et al., 2014). Overexpression of homologous and heterolo-gous sugar transporters enables recombinant strains to transport xylose, but have very limited positive effect on xylose fermentation and growth (Weierstall et al., 1999; Hamacher et al., 2002; Gardonyi et al., 2003; Sedlak and Ho, 2004; Saloheimo et al., 2007; Hector et al., 2008; Runquist et al., 2009). To improve xylose transporters, the substrate affinities for xylose of different yeast hexose transporters were altered and selected through mutagen-esis and screening approaches (Young et al., 2012, 2014; Farwick et al., 2014). These efforts identified regions and motifs of the hexose transporters as the engineering targets for reprograming transporter properties (Farwick et al., 2014; Young et al., 2014). However, whether the transport of xylose is the limiting factor for xylose fermentation requires more characterization. Theoretically, xylose uptake becomes a limiting step only when the rate of xylose fermentation is higher than xylose uptake (Cai et al., 2012). The wild-type S. cerevisiae CEN.PK2-1C with its native hexose transporter Hxt was reported to be able to take up 0.14 g xylose/h/g dry cell weight in the presence of 50 mM xylose, which exceeds the xylose consumption rate in most recombinant S. cerevisiae strains (Hamacher et al., 2002; Cai et al., 2012). Without optimization of sugar transporters, engineered yeast strains already achieved relatively high performance of xylose fermentation using native hexose sugar transporters for xylose uptake (Ho et al., 1998; Sonderegger et al., 2004). The potential beneficial effect of these improved xylose transporters in the recombinant yeast strains with high xylose metabolism remains to be tested.


Pretreatments such as dilute acid at elevated temperature are effective for the hydrolysis of pentose polymers in hemicellu-lose and also increase the access of cellulase enzymes to cellulose fibers. However, the fermentation of the resulting syrups, called hydrolyzates, is hindered by minor reaction products such as furan aldehydes including furfural and 5-hydroxymethylfurfural (5-HMF), organic acids, and phenolic compounds (Saha, 2003). Furfural and 5-HMF are formed by the dehydration of sugars (pentoses and hexoses, respectively) during pretreatment and more furfural than 5-HMF is present in most hemicellulose hydrolyzates (Saha, 2003; Geddes et al., 2010a,b, 2013). Furfural is of particular importance as a fermentation inhibitor because of its abundance and toxicity (Saha, 2003; Almeida et al., 2009; Mills et al., 2009; Geddes et al., 2010b, 2011). Furfural is more toxic than 5-HMF to industrial catalysts such as E. coli and S. cerevisiae (Zaldivar et al., 1999; Gorsich et al., 2006). In model studies with various hydrolyzate inhibitors, furfural was unique in potentiating the toxicity of other compounds (Zaldivar et al., 1999). The advancement of engineering tolerance to organic acids and phenolic compounds has been excellently summarized in recent reviews

(Mills et al., 2009; Laluce et al., 2012). This review mainly focuses on furan aldehydes as important lignocellulose inhibitors.

A significant amount of effort has been contributed to the identification and optimization of biological components to increase the resistance to furan aldehydes, especially furfural (Table 1). The toxicity mode of furan aldehydes is complex and involves multiple factors (Almeida et al., 2009; Lin et al., 2009a,b; Mills et al., 2009). Cellular growth is arrested in the presence of furan aldehydes and growth resumes after the complete reduction of furfural. This furan-induced delay in growth was observed in both E. coli and S. cerevisiae (Taherzadeh et al., 2000; Miller et al., 2009b; Wang et al., 2012b). There are two major metabolic pathways to metabolize or reduce furan aldehydes in nature (Figure 3). Some bacteria such as Cupriavidus basilensis HMF14 can catabolize furan aldehyde as a sole carbon source when growing aerobi-cally (Koopman et al., 2010). Furan aldehydes such as furfural are firstly oxidized into 2-furoic acid and then further metabolized to 2-oxoglutaric acid that eventually enters the TCA cycle to provide energy and biosynthetic building block (Trudgill, 1969; Koenig and Andreesen, 1990; Koopman et al., 2010) (Figure 3). The key step of this furfural degradation is dependent on oxygen thus limiting its application for anaerobic fermentative production

(Koopman et al., 2010; Ran et al., 2014). E. coli and S. cerevisiae do not have furan aldehydes oxidative degradation pathways. Under anaerobic fermentation conditions, these microbes use their native oxidoreductases to reduce furan aldehydes to furan alcohol, which is much less toxic (Zaldivar et al., 1999, 2000). Furan alcohols are secreted outside of cells and remain in the fermentation broth without further degradation (Liu and Blaschek, 2010; Wang et al., 2012b). Cells do not grow until furfural or 5-HMF is reduced to a low threshold concentration (~5 mM) (Liu and Blaschek, 2010; Wang et al., 2012b; Ran et al., 2014) (Figure 3). This native detoxification approach has been strengthened in S. cerevisiae strains by overexpression of the native oxidoreducase genes such as ADH1 (Laadan et al., 2008), ADH6 (Petersson et al., 2006; Almeida et al., 2008; Liu et al., 2008), and ADH7 (Liu et al., 2008) encoding the enzymes with activities to reduce furan aldehydes (Table 1). Overexpression of these oxidoreductase genes increases the 5-HMF reduction rate and shortens the lag time of cell growth. Interestingly, this native detoxification response causes the growth arrest in E. coli. The presence of furfural activates the expression of the yqhD gene encoding an oxidoreductase able to reduce furfural to furfuryl alcohol using NADPH as the reducing cofactor (Miller et al., 2009b; Turner et al., 2010). However, NADPH is essential

Table 1 | Beneficial genetic traits for furan aldehydes degradation and tolerance.

Beneficial genetic traits Microbial host

Proposed detoxification mechanism


yqhD deletion pntAB overexpression fucO overexpression Mutation of irrE ucpA overexpression thyA overexpression fucO missense mutations potE overexpression puuP overexpression plaP overexpression potABCD overexpression lpcA overexpression groESL overexpression ahpC overexpression yhiH overexpression rna overexpression dicA overexpression ZWF1 overexpression ADH6 overexpression ADH1 missense mutations

ADH7 overexpression YAP1 overexpression

Inactivation of SIZ1 Aerobic HMF degradation Aerobic furfural degradation

E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

S. cerevisiae S. cerevisiae S. cerevisiae

S. cerevisiae S. cerevisiae

S. cerevisiae C. basilensis HMF14 C. basilensis HMF14 P. putida Fu1, C. basilensis HMF14

Avoid the competition for NADPH Increase NADPH level Reduce furfural to furfuryl alcohol Stress related global regulator Unknown

Increase the availability of dTMP for DNA repair Improve furfural reductase activity

Polyamine binding to negatively charged cellular constituents

Polyamine binding to negatively charged cellular constituents

Polyamine binding to negatively charged cellular constituents

Polyamine binding to negatively charged cellular constituents

Strengthen cell wall or indirectly increase NADPH availability

Possibly related to solvent stress response





Maintain NADPH levels needed for furan oxidoreductases

Reduce HMF to alcohol form

S109P L116S, and Y294C increase affinity to NADH

Reduce HMF to alcohol form Mitigate oxidative stress

Likely related to oxidative stress Oxidize HMF Oxidize furfural

Miller et al. (2009b) Miller et al. (2009a) Wang etal. (2011) Wang etal. (2012a) Wang etal. (2012b) Zheng etal. (2012) Zheng etal. (2013) Geddes etal. (2014) Geddes etal. (2014) Geddes etal. (2014) Geddes etal. (2014) Glebes etal. (2014b) Glebes etal. (2014b) Glebes etal. (2014a) Glebes etal. (2014a) Glebes etal. (2014a) Glebes etal. (2014a) Gorsich etal. (2006) Petersson etal. (2006) Almeida et al. (2008), Laadan et al. (2008)

Liu etal. (2008)

Ma and Liu, 2010, Kim and Hahn, 2013

Xiao and Zhao, 2014

Koopman etal. (2010)

Trudgill, 1969, Koopman etal. (2010)

FIGURE 3 | Native furfural degradation pathways. There are two major native metabolic routes for furfural. In some Pseudomonas putida strains, using oxygen as the final electron acceptor furfural goes through a series of oxidation and eventually goes intoTCA cycle for further degradation. In contrast to aerobic degradation, the oxidoreductases with furfural reductase activity are recruited under anaerobic fermentation condition to reduce furfural to furfuryl alcohol, a less toxic product. Furfuryl alcohol is excreted into the medium.

for biosynthesis but is very limited under anaerobic xylose fermentation (Frick and Wittmann, 2005; Miller et al., 2009a). It is this depletion of NADPH by YqhD that has been proposed as the mechanism for growth inhibition in E. coli (Miller et al., 2009a,b; Turner et al., 2010). The NADPH-intensive pathway for sulfate assimilation was identified as a sensitive site that may be responsible for growth inhibition (Miller et al., 2009a). Addition of cysteine, deletion of yqhD, or increased expression of pntAB (transhydrogenase for interconversion of NADH and NADPH) conferred the tolerance to furan aldehydes including furfural and 5-HMF in E. coli (Miller et al., 2009a,b, 2010). To accelerate the furfural reduction but avoid using NADPH as the reducing cofactor, an alternative NADH-dependent furfural reductase is desired. A native oxidoreductase, FucO, was identified to have such properties and its overexpression did increase furfural tolerance in different E. coli biocatalysts (Wang et al., 2011). FucO normally functions in fucose metabolism and its catalytic efficiency for furfural reduction is low (Wang et al., 2011). The enzyme properties of FucO as a furfural reductase were improved by site-saturated mutagenesis and growth-based selection (Zheng et al., 2013). Overall, optimization of NADH-dependent furfural reductase has potential to shorten the lag phase and to increase tolerance of biocatalysts under fermentation conditions.

A variety of genomic and transcriptomic approaches have yielded many beneficial genetic traits related to furan aldehydes tolerance (Table 1). S. cerevisiae gene disruption library was screened for mutants with growth deficiencies in the presence of furfural and ZWF1 was found to relate to furfural tolerance (Gorsich et al., 2006). Overexpression of ZWF1 increased furfural tolerance (Gorsich et al., 2006). ZWF1 encodes glucose-6-phosphate dehydrogenase, which catalyzes the first step of the pentose phosphate pathway, the major pathway providing NADPH when utilizing glucose as the carbon source. A similar approach using genome-wide RNAi screen showed that inactivation of the SIZ1 gene increased furfural tolerance (Xiao and Zhao, 2014). SIZ1 encodes E3 SUMO-protein ligase and inactivation of SIZ1

increases the tolerance to oxidative stress besides furfural (Xiao and Zhao, 2014). At least part of the toxicity mechanism induced by furfural is suggested to be associated with oxidative stress (Mills et al., 2009). Furfural was shown to induce the accumulation of reactive oxygen species inside of the S. cerevisiae cells and to cause damage to mitochondria, vacuole membranes, and cytoskeletons (Allen et al., 2010). Furan aldehydes were also reported to act as thiol-reactive electrophiles, to directly activate Yap1 transcription factor and to deplete glutathione (Kim and Hahn, 2013). Overexpression of either wild-type YAP1 or its target genes CTA1 and CTT1encoding catalases increased tolerance to furan aldehydes (Kim and Hahn, 2013). Interestingly, furan aldehydes do not induce oxidative responses in E. coli. The expression of the genes in major oxidative regulons such as OxyR and SoxRS regulons is not activated by the presence of furfural (Miller et al., 2009a). This strain difference adds another layer of complexity to engineering tolerance of furan aldehydes. In E. coli, an oxidoreductase UcpA with an undefined function was found to be associated with furfural tolerance by a transcriptomic analysis and its overexpression increased furan aldehyde tolerance (Wang et al., 2012b). Genomic libraries from three different bacteria were screened for genes that conferred furfural resistance to E. coli on plates. Beneficial plasmids containing the thyA gene were recovered from all three genomic libraries. The thyA gene encodes thymidylate synthase, important for dTMP biosynthesis, suggesting furfural toxicity is possibly related to DNA damage (Zheng et al., 2012). The microarray studies and whole genome sequencing of furfural resistant E. coli mutants led to the discovery of some polyamine transporters including PotE, PuuP, PlaP, and PotABCD with a beneficial role for furfural tolerance (Geddes et al., 2014). The detoxification mechanism was proposed to relate to the protection role of polyamine for important cellular constituents such as DNA (Geddes et al., 2014). Other advanced genomic tools such as multiSCale Analysis of Library Enrichments (SCALE) (Lynch et al., 2007) and trackable multiplex recombineering (TRMR) (Warner et al., 2010) have been used to identify more furfural related genetic traits in E. coli (Glebes et al., 2014a,b). These experiments showed the lpcA, groESL, ahpC, yhiH, rna, and dicA genes are associated with furfural tolerance although the overexpression of these genes individually only showed limited positive effect (Glebes et al., 2014a,b). Another interesting approach is to select a mutant form of the stress-related exogenous regulator IrrE, which confers E. coli the tolerance to furan aldehydes (Wang et al., 2012a). Considering the complexity of the toxicity mode induced by furfural, it is not surprising to identify multiple biological parts beneficial for furan tolerance (Table 1). However, all these individual beneficial genetic traits discussed above only provide limited improvement for furan aldehyde tolerance. How to combine multiple beneficial genetic traits to achieve a significant increase of tolerance is a great challenge for synthetic biologists. An ideal synthetic detoxification system should contain a furfural responsive promoter driving the expression of the optimal combinations of different effector genes to minimize metabolic burden and maximize the benefit of effector genes (Figure 4).

There are at least two major challenges for designing such an integrated detoxification system. First, most epistatic interactions between beneficial genetic traits are not predictable and

No Furan aldehydes Off


Effector genes: A: Furfural reductase B: Anti-oxidative stress proteins C: Polyamine transporter D: Chaperonins

ho^j^o Furan aldehydes

-L- Â X B C":j b ■■

a Furan Detoxification cohort

FIGURE 4 |The integrated furan aldehydes detoxification system. A

furfural responsive promoter and multiple effector genes are integrated into the chromosome. In the absence of furan aldehydes, this artificial operon is inactive and effector genes are not expressed. Furan aldehydes activate the responsive promoter to drive the expression of effector genes. Effectors are produced to mitigate the toxicity of furan aldehydes. Example effectors shown in the graph are furfural reductase (A), anti-oxidative protein (B), polyamine transporter (C), and chaperonin (D), assuming these effectors have synergistic epistatic interaction. When furfural level decreases, promoter remains silenced and no more new effectors are made. This design provides a controllable mechanism for furfural tolerance to minimize metabolic burden and maximize the benefit of effector genes.

the experimental search for the optimal combination of multiple effector genes is time-consuming and labor-intensive (Sandoval et al., 2012b). Negative epistatic interactions are present for different beneficial genetic traits for furan aldehyde tolerance. For example, the combination of two beneficial traits, the increased expression of pntAB and the deletion of the yqhD gene together, made cells less tolerance to furfural than the cells with either one of these two beneficial genetic traits alone (Wang et al., 2013). Further characterization of the beneficial traits in a high-throughput manner is desired to eventually construct an optimal combination of multiple effector genes. Second, the technical challenges to achieve optimal expression of effector genes at the chromosomal level remain to be solved. The effector genes are normally expressed from an expression vector with expensive inducers and antibiotics or other selective conditions. The application of a plasmid-based expression system is undesired in large-scale bio-based production conditions due to the genetic instability, metabolic burden, and the costs (Keasling, 2008; Jarboe et al., 2010). Integration of furan aldehydes detoxification systems into the chromosome is desired. However, it is challenging to achieve the optimal expression of target genes at the chromosomal level, especially when high expression is needed.


Efficient xylose metabolism and tolerance to furan aldehydes are desired features of microbial catalysts used in lignocellulose conversion. Past efforts of synthetic biology focused on identification and optimization of individual biological parts needed for a successful lignocellulose conversion. We have gradually accumulated much knowledge about xylose metabolism and transport, glucose repression, and furan aldehyde toxicity. Limited success of lignocellulose conversion has been achieved using these individual optimized parts (Sandoval et al., 2012a; Wang et al., 2013). Instead of taking a reductionist approach, we are reaching a new phase to characterize the epistatic interactions and to integrate the optimal combinations of different biological parts. This development is dependent on the modular high-throughput approach for epistasis characterization and large-scale genome editing. With the new development of high-throughput techniques and genome editing tools such as CRISPR/Cas9 technology (Doench et al., 2014; Harrison et al., 2014; Sampson and Weiss, 2014), constructing an effective platform strain for lignocellulose conversion is in the scope. The platform strains with high efficiency of sugar co-utilization and tolerance to chemical insult can be used to produce a variety of fuels and chemicals from lignocellulosic biomass by metabolic engineering. These common platforms can also be tuned to different types of biomass by laboratory adaptive evolution.


This work was supported by start-up fund from Arizona State University.


Allen, S. A., Clark, W., McCaffery, J. M., Cai, Z., Lanctot, A., Slininger, P. J., et al. (2010). Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnol. Biofuels 3, 2. doi:10.1186/1754-6834-3-2

Almeida, J. R., Bertilsson, M., Gorwa-Grauslund, M. F., Gorsich, S., and Liden, G. (2009). Metabolic effects of furaldehydes and impacts on biotechnologi-cal processes. Appl. Microbiol. Biotechnol. 82,625-638. doi:10.1007/s00253-009-1875-1

Almeida, J. R., Roder, A., Modig, T., Laadan, B., Liden, G., and Gorwa-Grauslund, M. F. (2008). NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 78, 939-945. doi:10.1007/s00253-008-1364-y. Alvira, P., Tomas-Pejo, E., Ballesteros, M., and Negro, M. J. (2010). Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851-4861. doi:10.1016/j.biortech. 2009.11.093

Bacchus, W., Lang, M., El-Baba, M. D., Weber, W., Stelling, J., and Fussenegger M. (2012). Synthetic two-way communication between mammalian cells. Nat. Biotechnol. 30,991-996. doi:10.1038/nbt.2351 Bettiga,M.,Hahn-Hagerdal,B., and Gorwa-Grauslund, M. F. (2008). Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabi-nose and xylose fermenting Saccharomyces cerevisiae strains. Biotechnol. Biofuels 1,16. doi:10.1186/1754-6834-1-16 Bommarius, A. S., Sohn, M., Kang, Y., Lee, J. H., and Realff, M. J. (2014). Protein engineering of cellulases. Curr. Opin. Biotechnol. 29C, 139-145. doi:10.1016/j. copbio.2014.04.007

Brat, D., Boles, E., and Wiedemann, B. (2009). Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 75, 2304-2311. doi:10.1128/AEM.02522- 08 Cai, Z., Zhang, B., and Li, Y. (2012). Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives. Biotechnol. J. 7, 34-46. doi:10.1002/biot.201100053

Cameron, D. E., Bashor, C. J., and Collins, J. J. (2014). A brief history of synthetic

biology. Nat. Rev. Microbiol. 12, 381-390. doi:10.1038/nrmicro3239 Chiang, C. J., Lee, H. M., Guo, H. J., Wang, Z. W., Lin, L. J., and Chao, Y. P. (2013). Systematic approach to engineer Escherichia coli pathways for co-utilization of a glucose-xylose mixture. J. Agric. Food Chem. 61, 7583-7590. doi:10.1021/jf401230r Chu, B. C., and Lee, H. (2007). Genetic improvement of Saccharomyces cerevisiae for xylose fermentation. Biotechnol. Adv. 25, 425-441. doi:10.1016/j.biotechadv. 2007.04.001

Conway, T. (1992). The Entner-Doudoroff pathway: history, physiology and molecular biology. FEMS Microbiol. Rev. 9, 1-27. doi:10.1111/j.1574-6968.1992. tb05822.x

Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., et al. (2014).RationaldesignofhighlyactivesgRNAsforCRISPR-Cas9-mediatedgene inactivation. Nat. Biotechnol. 32, 1262-1267. doi:10.1038/nbt.3026 Elkins, J. G., Raman, B., and Keller, M. (2010). Engineered microbial systems for enhanced conversion of lignocellulosic biomass. Curr. Opin. Biotechnol. 21, 657-662. doi:10.1016/j.copbio.2010.05.008 Farwick, A., Bruder, S., Schadeweg, V., Oreb, M., and Boles, E. (2014). Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose. Proc. Natl. Acad. Sci. U.S.A. 111,5159-5164. doi:10.1073/pnas.1323464111 Feldmann, S. D., Sahm, H., and Sprenger, G. A. (1992). Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains. Appl. Microbiol. Biotechnol. 38, 354-361. doi:10.1007/BF00170086 Frick, O.,andWittmann, C. (2005). Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis. Microb. Cell Fact. 4, 30. doi:10.1186/1475-2859-4-30 Galazka, J. M., Tian, C. G., Beeson, W. T., Martinez, B., Glass, N. L., and Cate, J. H. D. (2010). Cellodextrin transport in yeast for improved biofuel production. Science 330,84-86. doi:10.1126/science.1192838 Gardonyi, M., and Hahn-Hagerdal, B. (2003). The Streptomyces rubiginosus xylose isomerase is misfolded when expressed in Saccharomyces cerevisiae. Enzyme Microb. Technol. 32, 252-259. doi:10.1016/S0141-0229(02)00285-5 Gardonyi, M., Jeppsson, M., Liden, G., Gorwa-Grausland, M. F., and Hahn-Hagerdal, B. (2003). Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae. Biotechnol. Bioeng. 82, 818-824. doi:10. 1002/bit.10631

Garvey, M., Klose, H., Fischer, R., Lambertz, C., and Commandeur, U. (2013). Cel-lulases for biomass degradation: comparing recombinant cellulase expression platforms. Trends Biotechnol. 31, 581-593. doi:10.1016/j.tibtech.2013.06.006 Geddes, C. C., Mullinnix, M. T., Nieves, I. U., Hoffman, R. W., Sagues, W. J., York, S. W., et al. (2013). Seed train development for the fermentation of bagasse from sweet sorghum and sugarcane using a simplified fermentation process. Bioresour. Technol. 128, 716-724. doi:10.1016/j.biortech.2012.09.121 Geddes, C. C., Mullinnix, M. T., Nieves, I. U., Peterson, J. J., Hoffman, R. W., York, S. W., et al. (2010a). Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160. Bioresour. Technol. 102,2702-2711. doi:10.1016/j.biortech.2010.10.143 Geddes, C. C., Peterson, J. J., Roslander, C., Zacchi, G., Mullinnix, M. T., Shanmugam, K. T., et al. (2010b). Optimizing the saccharification of sugar cane bagasse using dilute phosphoric acid followed by fungal cellulases. Bioresour. Technol. 101, 1851-1857. doi:10.1016/j.biortech.2009.09.070 Geddes, C. C., Nieves, I. U., and Ingram, L. O. (2011). Advances in ethanol production. Curr. Opin. Biotechnol. 22,312-319. doi:10.1016/j.copbio.2011.04.012 Geddes, R. D., Wang, X., Yomano, L. P., Miller, E. N., Zheng, H., Shanmugam, K. T., et al. (2014). Polyamine transporters and polyamines increase furfural tolerance during xylose fermentation with ethanologenic Escherichia coli strain LY180. Appl. Environ. Microbiol. 80, 5955-5964. doi:10.1128/AEM.01913-14 Girio, F. M., Fonseca, C., Carvalheiro, F., Duarte, L. C., Marques, S., and Bogel-Lukasik, R. (2010). Hemicelluloses for fuel ethanol: a review. Bioresour. Technol. 101,4775-4800. doi:10.1016/j.biortech.2010.01.088 Glebes, T. Y., Sandoval, N. R., Gillis, J. H., and Gill, R. T. (2014a). Comparison of genome-wide selection strategies to identify furfural tolerance genes in Escherichia coli. Biotechnol. Bioeng. 112,129-140. doi:10.1002/bit.25325 Glebes, T. Y., Sandoval, N. R., Reeder, P. J., Schilling, K. D., Zhang, M., and Gill, R. T. (2014b). Genome-wide mapping of furfural tolerance genes in Escherichia coli. PLoS ONE 9:e87540. doi:10.1371/journal.pone.0087540 Gorsich, S. W., Dien, B. S., Nichols, N. N., Slininger, P. J., Liu, Z. L., and Skory, C. D. (2006). Tolerance to furfural-induced stress is associated with pentose phosphate

pathway genes ZWF1, GND1,RPE1,and TKL1 in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 71, 339-349. doi:10.1007/s00253-005-0142-3 Ha, S. J., Galazka, J. M., Kim, S. R., Choi, J. H., Yang, X. M., Seo, J. H., et al. (2011). Engineered Saccharomyces cerevisiae capable of simultaneous cel-lobiose and xylose fermentation. Proc. Natl. Acad. Sci. U.S.A. 108, 504-509. doi:10.1073/pnas.1010456108 Hamacher, T., Becker, J., Gardonyi, M., Hahn-Hagerdal, B., and Boles, E. (2002). Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148, 2783-2788. Harrison, M. M., Jenkins, B. V., O'Connor-Giles, K. M., and Wildonger, J. (2014). A CRISPR view of development. Genes Dev. 28, 1859-1872. doi:10.1101/gad. 248252.114

Hasunuma, T., Okazaki, F., Okai, N., Hara, K. Y., Ishii, J., and Kondo, A. (2013). A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology. Bioresour. Technol. 135, 513-522. doi:10.1016/j.biortech.2012.10.047 Hector, R. E., Qureshi, N., Hughes, S. R., and Cotta, M. A. (2008). Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption. Appl. Microbiol. Biotech-nol. 80, 675-684. doi:10.1007/s00253-008-1583-2 Ho, N. W., Chen, Z., and Brainard, A. P. (1998). Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl. Environ. Microbiol. 64, 1852-1859. Jarboe, L. R., Grabar, T. B., Yomano, L. P., Shanmugan, K. T., and Ingram, L. O. (2007). Development of ethanologenic bacteria. Adv. Biochem. Eng. Biotechnol. 108,237-261.

Jarboe, L. R., Zhang, X.,Wang,X., Moore, J. C., Shanmugam, K. T., and Ingram, L. O. (2010). Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology. J. Biomed. Biotechnol. 2010, 761042. doi:10.1155/2010/761042 Jeffries, T. W. (1983). Utilization of xylose by bacteria, yeasts, and fungi. Adv.

Biochem. Eng. Biotechnol. 27, 1-32. Jeffries, T. W., and Jin, Y. S. (2004). Metabolic engineering for improved fermentation of pentoses by yeasts. Appl. Microbiol. Biotechnol. 63, 495-509. doi:10.1007/s00253-003-1450-0 Jin, Y. S., Alper, H., Yang, Y. T., and Stephanopoulos, G. (2005). Improvement of xylose uptake and ethanol production in recombinant Saccharomyces cerevisiae through an inverse metabolic engineering approach. Appl. Environ. Microbiol. 71,8249-8256. doi:10.1128/AEM.71.12.8249-8256.2005 Keasling, J. D. (2008). Synthetic biology for synthetic chemistry. ACS Chem. Biol. 3,

64-76. doi:10.1021/cb7002434 Kim, D., and Hahn, J. S. (2013). Roles of the Yap1 transcription factor and antioxidants in Saccharomyces cerevisiae's tolerance to furfural and 5-hydroxymethylfurfural, which function as thiol-reactive electrophiles generating oxidative stress. Appl. Environ. Microbiol. 79, 5069-5077. doi:10.1128/AEM. 00643-13

Kim, J. H., Block, D. E., and Mills, D. A. (2010). Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass. Appl. Microbiol. Biotechnol. 88,1077-1085. doi:10.1007/s00253-010-2839-1 Kim, S. R., Park, Y. C., Jin, Y. S., and Seo, J. H. (2013). Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnol. Adv. 31, 851-861. doi:10.1016/j.biotechadv.2013.03.004 Koenig, K., and Andreesen, J. R. (1990). Xanthine dehydrogenase and 2-furoyl-coenzyme-A dehydrogenase from Pseudomonas putida Ful - 2 molybdenum-containing dehydrogenases of novel structural composition. J. Bacteriol. 172, 5999-6009.

Koopman, F., Wierckx, N., De Winde, J. H., and Ruijssenaars, H. J. (2010). Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14. Proc. Natl. Acad. Sci. U.S.A. 107,4919-4924. doi:10.1073/pnas.0913039107 Kotter, P., Amore, R., Hollenberg, C. P., and Ciriacy, M. (1990). Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr. Genet. 18, 493-500. doi:10.1007/BF00327019 Kuyper, M., Harhangi, H. R., Stave, A. K., Winkler, A. A., Jetten, M. S., De Laat, W. T., et al. (2003). High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae? FEMS Yeast Res. 4, 69-78. doi:10.1016/S1567-1356(03)00141-7

Laadan, B., Almeida, J. R., Radstrom, P., Hahn-Hagerdal, B., and Gorwa-Grauslund, M. (2008). Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol dehydrogenase in Saccharomyces cerevisiae. Yeast 25, 191-198. doi:10.1002/yea.1578 Laluce, C., Schenberg, A. C. G., Gallardo, J. C. M., Coradello, L. F. C., and Pombeiro-Sponchiado, S. R. (2012). Advances and developments in strategies to improve strains of Saccharomyces cerevisiae and processes to obtain the lignocellulosic ethanol - a review. Appl. Biochem. Biotechnol. 166, 1908-1926. doi:10.1007/s12010-012-9619-6 Lee,K. H., and Kim,D. M. (2013). Applications of cell-free protein synthesis in synthetic biology: interfacing bio-machinery with synthetic environments. Biotechnol. J. 8,1292-1300. doi:10.1002/biot.201200385 Lee, S. M., Jellison, T., and Alper, H. S. (2012). Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae. Appl. Environ. Microbiol. 78, 5708-5716. doi:10.1128/AEM.01419-12 Lin, F. M., Qiao, B., and Yuan, Y. J. (2009a). Comparative proteomic analysis of tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to furfural, a lignocellulosic inhibitorycompound. Appl. Environ. Microbiol. 75, 3765-3776. doi:10.1128/AEM.02594-08 Lin, F. M., Tan, Y., and Yuan, Y. J. (2009b). Temporal quantitative proteomics of Saccharomyces cerevisiae in response to a nonlethal concentration of furfural. Proteomics 9, 5471-5483. doi:10.1002/pmic.200900100 Liu, C. Q., Goodman, A. E., and Dunn, N. W. (1988). Expression of cloned xan-thomonas D-xylose catabolic genes in Zymomonas mobilis. J. Biotechnol. 7,61-70. doi:10.1016/0168-1656(88)90035-1 Liu, Z. L., and Blaschek, H. P. (2010). "Biomass converion inhibitors and in situ detoxification," in Biomass to Biofuels: Strategies for Global Industries, eds O. A. Vertès, H. P. Blaschek, and H. Yukawa (West Sussex: John Wiley and Sons), 233-259.

Liu,Z. L., Moon, J., Andersh, B. J., Slininger, P. J., and Weber, S. (2008). Multiple genemediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cere-visiae. Appl. Microbiol. Biotechnol. 81,743-753. doi:10.1007/s00253-008-1702-0 Liu,Z. L., Slininger, P. J., Dien, B. S.,Berhow, M. A., Kurtzman, C. P., and Gorsich, S. W. (2004). Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J. Ind. Microbiol. Biotechnol. 31, 345-352. Liu, Z. L., Slininger, P. J., and Gorsich, S. W. (2005). Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. Appl. Biochem. Biotechnol. 12,451-460. doi:10.1385/ABAB:121:1-3:0451 Luhe, A. L., Lim, C. Y., Gerken, H.,Wu, J., and Zhao, H. (2014). Furfural and hydrox-ymethylfurfural tolerance in Escherichia coli DeltaacrR regulatory mutants. Biotechnol. Appl. Biochem. doi:10.1002/bab.1232 Luo, Y., Zhang, T., and Wu, H. (2014). The transport and mediation mechanisms of the common sugars in Escherichia coli. Biotechnol. Adv. 32, 905-919. doi: 10.1016/j.biotechadv.2014.04.009 Lynch, M. D., Warnecke, T., and Gill, R. T. (2007). SCALEs: multiscale analysis of

library enrichment. Nat. Methods 4, 87-93. doi:10.1038/nmeth946 Lynd, L. R. (1990). Large-scale fuel ethanol from lignocellulose - potential, economics, and research priorities. Appl. Biochem. Biotechnol. 2, 695-719. doi:10.1007/ BF02920289

Ma, M., and Liu,Z. L. (2010). Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Sac-charomyces cerevisiae. BMC Genomics 11:660. doi: 10.1186/1471-2164-11-660 Madhavan, A., Tamalampudi, S., Ushida, K., Kanai, D., Katahira, S., Srivas-tava, A., et al. (2009). Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol. Appl. Microbiol. Biotechnol. 82, 1067-1078. doi:10.1007/s00253-008-1794-6 Martinez, A., Rodriguez, M. E., York, S. W., Preston, J. F., and Ingram, L. O. (2000). Effects of Ca(OH)(2) treatments ("overliming") on the composition and toxicity of bagasse hemicellulose hydrolysates. Biotechnol. Bioeng. 69, 526-536. doi:10.1002/1097-0290(20000905)69:5<526::AID-BIT7>3.0.C0;2-E Matsushika, A., Inoue, H., Kodaki, T., and Sawayama, S. (2009). Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives. Appl. Microbiol. Biotechnol. 84, 37-53. doi:10.1007/s00253-009-2101-x

Miller, E. N., Jarboe, L. R., Turner, P. C., Pharkya, P., Yomano, L. P., York, S. W., et al. (2009a). Furfural inhibits growth by limiting sulfur assimilation in ethanolo-genic Escherichia coli strain LY180. Appl. Environ. Microbiol. 75, 6132-6141. doi:10.1128/AEM.01187-09 Miller, E. N., Jarboe, L. R., Yomano, L. P., York, S. W., Shanmugam, K. T., and Ingram, L. O. (2009b). Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli. Appl. Environ. Microbiol. 75,4315-4323. doi:10.1128/AEM.00567-09 Miller, E. N., Turner, P. C., Jarboe, L. R., and Ingram, L. O. (2010). Genetic changes that increase 5-hydroxymethyl furfural resistance in ethanol-producing Escherichia coli LY180. Biotechnol. Lett. 32, 661-667. doi:10.1007/s10529-010-0209-9

Mills, T. Y., Sandoval, N. R., and Gill, R. T. (2009). Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnol. Biofuels 2, 26. doi:10.1186/1754-6834-2-26 Moses, T., Pollier, J., Thevelein, J. M., and Goossens, A. (2013). Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology invivo and invitro. New Phytol. 200, 27-43. doi:10.1111/nph.12325 Nissen, T. L., Anderlund, M., Nielsen, J., Villadsen, J., and Kielland-Brandt, M. C. (2001). Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool. Yeast 18,19-32. doi:10.1002/1097-0061(200101)18:1<19::AID-YEA650>3.3.C0;2-X Petersson, A., Almeida, J. R. M., Modig, T., Karhumaa, K., Hahn-Hagerdal, B., Gorwa-Grauslund, M. F., et al. (2006). A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance. Yeast 23,455-464. doi:10.1002/yea.1370 Ramseier, T. M. (1996). Cra and the control of carbon flux via metabolic pathways.

Res. Microbiol. 147, 489-493. doi:10.1016/0923-2508(96)84003-4 Ran, H., Zhang, J., Gao, Q. Q., Lin, Z. L., and Bao, J. (2014). Analysis of biodegradation performance of furfural and 5-hydroxymethylfurfural by Amorphotheca resinae ZN1. Biotechnol. Biofuels 7,51. doi:10.1186/1754-6834-7-51 Richard, P., Toivari, M. H., and Penttila, M. (2000). The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism. FEMS Microbiol. Lett. 190, 39-43. doi:10.1111/j.1574-6968.2000.tb09259.x Rizzi, M., Erlemann, P., Buithanh, N. A., and Dellweg, H. (1988). Xylose fermentation by yeasts: 4. Purification and kinetic-studies of xylose reductase from Pichia stipitis. Appl. Microbiol. Biotechnol. 29, 148-154. doi:10.1007/BF00939299 Rizzi, M., Klein, C., Schulze, C., Bui-Thanh, N. A., and Dellweg, H. (1989). Xylose fermentation by yeasts. 5. Use of ATP balances for modeling oxygen-limited growth and fermentation of yeast Pichia stipitis with xylose as carbon source. Biotechnol. Bioeng. 34, 509-514. doi:10.1002/bit.260340411 Runquist, D., Fonseca, C., Radstrom, P., Spencer-Martins, I., and Hahn-Hagerdal, B. (2009). Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cere-visiae. Appl. Microbiol. Biotechnol. 82,123-130. doi:10.1007/s00253-008-1773-y Saha, B. C. (2003). Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30,

279-291. doi:10.1007/s10295-003-0049-x Saloheimo, A., Rauta, J., Stasyk, O. V., Sibirny, A. A., Penttila, M., and Ruohonen, L. (2007). Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases. Appl. Microbiol. Biotechnol. 74, 1041-1052. doi:10.1007/s00253-006-0747-1 Sampson, T. R., and Weiss, D. S. (2014). Exploiting CRISPR/Cas systems for biotechnology. Bioessays 36, 34-38. doi:10.1002/bies.201300135 Sandoval, N. R., Kim, J. Y., Glebes, T. Y., Reeder, P. J., Aucoin, H. R., Warner, J. R., et al. (2012a). Strategy for directing combinatorial genome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 109,10540-10545. doi:10.1073/pnas. 1206299109

Sandoval, N. R., Kim, J. Y. H., Glebes, T. Y., Reeder, P. J., Aucoin, H. R., Warner, J. R., et al. (2012b). Strategy for directing combinatorial genome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 109, 10540-10545. doi:10.1073/ pnas.1206299109

Sarthy, A. V., McConaughy, B. L., Lobo, Z., Sundstrom, J. A., Furlong, C. E., and Hall, B. D. (1987). Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 53, 1996-2000. Sedlak, M., and Ho, N. W. Y. (2004). Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast. Yeast 21, 671-684. doi:10. 1002/yea.1060

Service, R. F. (2007). Cellulosic ethanol - biofuel researchers prepare to reap a new

harvest. Science 315, 1488-1491. doi:10.1126/science.315.5818.1488 Sheridan, C. (2013). Big oil turns on biofuels. Nat. Biotechnol. 31, 870-873.

doi:10.1038/nbt.2704 Skotnicki, M. L., Warr, R. G., Goodman, A. E., Lee, K. J., and Rogers, P. L. (1983). High-productivity alcohol fermentations using Zymomonas mobilis. Biochem. Soc. Symp. 48, 53-86. Sonderegger, M., Jeppsson, M., Larsson, C., Gorwa-Grauslund, M. F., Boles, E., Olsson, L., et al. (2004). Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains. Biotechnol. Bioeng. 87,90-98. doi:10.1002/bit.20094 Sousa, L. D., Chundawat, S. P. S., Balan, V., and Dale, B. E. (2009). 'Cradle-to-grave' assessment of existing lignocellulose pretreatment technologies. Curr. Opin. Biotechnol 20, 339-347. doi:10.1016/j.copbio.2009.05.003 Taherzadeh, M. J., Gustafsson, L., Niklasson, C., and Liden, G. (2000). Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol 53, 701-708. doi:10.1007/s002530000328 Tantirungkij, M., Seki, T., and Yoshida, T. (1994). Genetic improvement of Saccharomyces cerevisiae for ethanol production from xylose. Ann. N. Y. Acad. Sci. 721, 138-147. doi:10.1111/j.1749-6632.1994.tb47386.x Toivari, M. H., Salusjarvi, L., Ruohonen, L., and Penttila, M. (2004). Endogenous xylose pathway in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 70, 3681-3686. doi:10.1128/AEM.70.6.3681-3686.2004 Traff, K. L., Jonsson, L. J., and Hahn-Hagerdal, B. (2002). Putative xylose and ara-binose reductases in Saccharomyces cerevisiae. Yeast 19,1233-1241. doi:10.1002/ yea.913

Trantidou, T., Rao, C., Barrett, H., Camelliti, P., Pinto, K., Yacoub, M. H., et al. (2014). Selective hydrophilic modification of Parylene C films: a new approach to cellmicro-patterningforsyntheticbiologyapplications.Biofabrication6:025004. doi:10.1088/1758-5082/6/2/025004 Trudgill, P. W. (1969). Metabolism of 2-furoic acid by Pseudomonas F2. Biochem. J. 113,577-587.

Turner, P. C., Miller, E. N., Jarboe, L. R., Baggett, C. L., Shanmugam, K. T., and Ingram, L. O. (2010). YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance. J. Ind. Microbiol. Biotechnol 38,431-439. doi:10.1007/s10295-10010-10787-10295 Walfridsson, M.,Bao,X. M.,Anderlund, M.,Lilius, G.,Bulow, L., and Hahnhagerdal, B. (1996). Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase. Appl. Environ. Microbiol. 62, 4648-4651. Wang, J. Q., Zhang, Y., Chen, Y. L., Lin, M., and Lin, Z. L. (2012a). Global regulator engineering significantly improved Escherichia coli tolerances toward inhibitors of lignocellulosic hydrolysates. Biotechnol. Bioeng. 109, 3133-3142. doi:10.1002/bit.24574 Wang, X., Miller, E. N., Yomano, L. P., Shanmugam, K. T., and Ingram, L. O. (2012b). Increased furan tolerance in Escherichia coli due to a cryptic ucpA gene. Appl. Environ. Microbiol. 78, 2452-2455. doi:10.1128/AEM.07783-11 Wang, X., Miller, E. N., Yomano, L. P., Zhang, X., Shanmugam, K. T., and Ingram, L. O. (2011). Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate. Appl. Environ. Microbiol. 77, 5132-5140. doi:10.1128/AEM.05008-11 Wang, X., Yomano, L. P., Lee, J. Y., York, S. W., Zheng, H., Mullinnix, M. T., et al. (2013). Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals. Proc. Natl. Acad. Sci. U.S.A. 110,4021-4026. doi:10.1073/pnas.1217958110 Warner, J. R., Reeder, P. J., Karimpour-Fard, A., Woodruff, L. B. A., and Gill, R. T. (2010). Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nat. Biotechnol. 28, 856-U138. doi:10.1038/nbt.1653 Way, J. C., Collins, J. J., Keasling, J. D., and Silver, P. A. (2014). Integrating biological redesign: where synthetic biology came from and where it needs to go. Cell 157, 151-161. doi:10.1016/j.cell.2014.02.039

Weierstall, T., Hollenberg, C. P., and Boles, E. (1999). Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis. Mol. Microbiol. 31, 871-883. doi:10.1046/j.1365-2958.1999.01224.x Xiao, H., and Zhao, H. (2014). Genome-wide RNAi screen reveals the E3 SUMO-protein ligase gene SIZ1 as a novel determinant of furfural tolerance in Saccharomyces cerevisiae. Biotechnol. Biofuels 7, 78. doi:10.1186/1754-6834-7-78 Xu, P., Bhan, N., and Koffas, M. A. G. (2013). Engineering plant metabolism into microbes: from systems biology to synthetic biology. Curr. Opin. Biotechnol. 24, 291-299. doi:10.1016/j.copbio.2012.08.010 Yang, V. W., and Jeffries, T. W. (1997). Regulation of phosphotransferases in glucose- and xylose-fermenting yeasts. Appl. Biochem. Biotechnol. 6, 97-108. doi:10.1007/BF02920416 Yomano, L. P., York, S.W., Shanmugam, K. T., and Ingram, L. O. (2009). Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli. Biotechnol. Lett. 31,1389-1398. doi:10.1007/s10529-009-0011-8

Young, E., Lee, S. M., and Alper, H. (2010). Optimizing pentose utilization in yeast: the need for novel tools and approaches. Biotechnol. Biofuels 3, 24. doi:10.1186/1754-6834-3-24 Young, E. M., Comer, A. D., Huang, H. S., and Alper, H. S. (2012). A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae. Metab. Eng. 14, 401-411. doi:10.1016/j.ymben.2012.03.004 Young, E. M., Tong, A., Bui, H., Spofford, C., and Alper, H. S. (2014). Rewiring yeast sugar transporter preference through modifying a conserved protein motif. Proc. Natl. Acad. Sci. U.S.A. 111,131-136. doi:10.1073/pnas.1311970111 Zaldivar, J., Martinez, A., and Ingram, L. O. (1999). Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol. Bioeng. 65,24-33. doi:10.1002/(SICI)1097-0290(19991005)65:1<24::AID-BIT4>3. 0.C0;2-2

Zaldivar, J., Martinez, A., and Ingram, L. O. (2000). Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanolo-genic Escherichia coli. Biotechnol. Bioeng. 68, 524-530. doi:10.1002/(SICI)1097-0290(20000605)68:5<524::AID-BIT6>3.3.CO;2-K Zhang, M., Eddy, C., Deanda, K., Finkestein, M., and Picataggio, S. (1995). Metabolic engineering of apentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267, 240-243. doi:10.1126/science.267.5195.240 Zheng, H., Wang, X., Yomano, L. P., Geddes, R. D., Shanmugam, K. T., and Ingram, L. O. (2013). Improving Escherichia coli FucO for furfural tolerance by saturation mutagenesis of individual amino acid positions. Appl. Environ. Microbiol. 79,3202-3208. doi:10.1128/AEM.00149-13 Zheng, H., Wang, X., Yomano, L. P., Shanmugam, K. T., and Ingram, L. O. (2012). Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA. Appl. Environ. Microbiol. 78, 4346-4352. doi:10.1128/AEM.00356-12

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Received: 26 November 2014; accepted: 04 February 2015; published online: 18 February 2015.

Citation: Nieves LM, Panyon LA and Wang X (2015) Engineering sugar utilization and microbial tolerance toward lignocellulose conversion. Front. Bioeng. Biotechnol. 3:17. doi: 10.3389/fbioe.2015.00017

This article was submitted to Synthetic Biology, a section of the journal Frontiers in Bioengineering and Biotechnology.

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