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Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes Tomoko MAEHARAa, Koji TAKABATAKEb, Satoshi KANEKOa,* a
Food Biotechnology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan b Forest Institute, Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, 3 Yoshimine, Tateyama, Toyama 930-1362, Japan
article info
abstract
Article history:
To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isom-
Received 18 March 2013
erase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the
Received in revised form
gene expression was evaluated in transformants. mRNA expression of the putative XI gene
4 September 2013
and XI activity were observed in two transformants, indicating that the putative gene from
Accepted 30 September 2013
A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, eth-
Available online 12 October 2013
anol production from xylose was increased in the recombinant strains. This is the first re-
Corresponding Editor:
port demonstrating the possibility of using plant genes as candidates for improving the
Daniel Eastwood
characteristics of F. velutipes. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Basidiomycete fungi Ethanol fermentation Flammulina velutipes Heterologous gene expression Xylose metabolism
Introduction To achieve an economic bioethanol production from lignocellulosic biomass, consolidated bioprocessing (CBP) that combines enzyme production, enzymatic saccharification and ethanol fermentation in one step was proposed by Lynd et al. (2005). Recently, several studies of CBP capable of high yield conversion of biomass to ethanol were reported using the combined abilities of two organisms such as a cellulolytic enzyme-producing organism and an ethanol-producing organism (Zhang & Zhang 2010; van Zyl et al. 2011). However, no single organism suitable for CBP has been developed.
Flammulina velutipes is a wood-rotting fungus that can completely degrade lignocellulose. Because the F. velutipes Fv-1 strain produces both ethanol and cellulolytic enzymes, we investigated CBP bioethanol production using F. velutipes and found that this strain was a good candidate for ethanol production from lignocelluloses by CBP (Mizuno et al. 2009a, 2009b; Maehara et al. 2013). Furthermore, we have developed gene transformation methods for F. velutipes (Maehara et al. 2010a, 2010b). The Fv-1 strain was able to ferment D-glucose, D-fructose, D-mannose, sucrose, maltose, cellobiose and cellulose; however, it can hardly produce ethanol from pentoses (Maehara et al. 2013). Because lignocellulosic biomass contains
* Corresponding author. Tel.: þ81 29 838 8063; fax: þ81 29 838 7996. E-mail address: [email protected] (S. Kaneko). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.09.005
Heterologous gene expression in Flammulina velutipes for ethanol production
up to approximately 30e40% pentoses, utilisation of pentoses is necessary for effective ethanol production. Therefore, improvement of pentose fermentation by F. velutipes would be required. A major bioethanol producer, Saccharomyces cerevisiae is not able to ferment pentoses. Thus, many studies have been conducted for the genetic engineering of this yeast directed at improving pentose fermentation (Jeffries & Jin 2004; Matsushika et al. 2009). There have been two general approaches to this work. The first approach has been the expression of heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes to improve the endogenous pentose-metabolising pathway. Recombinant S. cerevisiae strains that expressed XR and XDH showed improved ethanol production from D-xylose. However, xylitol accumulation caused by an NADPH/NAD cofactor imbalance under anaerobic condition was observed; thus, it would be desirable to introduce additional enzyme-encoding genes to address this problem. The second approach has been the expression of a heterologous xylose isomerase (XI) gene to produce a new bypass pathway for pentose metabolism to convert D-xylose to D-xylulose directly. Because XI does not require redox cofactors, it does not cause xylitol accumulation and cofactor imbalances; however, in general, XI is involved in the pentose pathway of bacteria and is difficult to express in eukaryotes. Because F. velutipes is expected to have the same sugar metabolism pathway as S. cerevisiae (Barnett 1976; Maehara et al. 2013) and because the introduction of multiple enzymeencoding genes into F. velutipes would be difficult, we aimed to introduce a heterologous XI gene from a eukaryotic organism into F. velutipes. In this study, we focused on the gene encoding a putative XI from the plant Arabidopsis thaliana (AtXI ). This putative gene was transformed into the F. velutipes Fv-1 strain, and its expression and effect on ethanol production from D-xylose were evaluated in F. velutipes. To our knowledge, there is no report on the use of a plant XI gene for improving pentose fermentation and our study is the first report on improving the characteristics of F. velutipes by genetic engineering.
Materials and methods Strains, media and culture conditions The wood-rotting fungus Flammulina velutipes Fv-1 strain was used as the transformation host. The strain was grown at 25 C with shaking at 120 rpm in B medium [1% yeast extract, 1% peptone, 1% D-glucose, 0.1% KH2PO4 and 0.01% MgSO4$7H2O (pH 5.5)]. Hygromycin B (30 mg ml1) was added to the cultivation medium if required. The bacterial strain Escherichia coli DH5a was used for cloning AtXI. The E. coli strain was grown at 37 C in LuriaeBertani (LB) medium. Ampicillin (100 mg ml1) was added to the medium when required. The seeds of Arabidopsis thaliana were cultivated at 20 C on MurashigeeSkoog medium supplemented with 2% sucrose, vitamin solution (0.3% thiamine HCl, 0.5% nicotinic acid and 0.05% pyridoxine HCl) and 0.3% agar.
Plasmid construction The plasmid used in this study is shown in Fig 1. The plasmid designated pFvGX carried a 1368-bp XI gene fragment isolated
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Fig 1 e Structures of the plasmid used in this study. pFvGX (7828 bp) was derived from pFvG (Maehara et al. 2010a) by cloning the AtXI gene without a signal sequence into the multi-cloning sites (MCS) of pFvG. hph is a hygromycin B phosphotransferase gene and ampr is an ampicillin resistance gene. gpd is glyceraldehydes-3-phosphate dehydrogenase and trp1 is the tryptophan synthetase gene.
from Arabidopsis thaliana. Total RNA of A. thaliana was prepared using RNeasy Plant Mini Kit (QIAGEN, CA, USA), and cDNA was prepared by reverse transcriptase (RT) reactions using ReverTra Ace-a (Toyobo, Osaka, Japan) with 0.1 ng of total RNA as the template. The gene encoding XI from A. thaliana was amplified by PCR with KOD-plus- ver.2 (Toyobo, Osaka, Japan) using the primer pairs 50 -GATCCACCAACATGTCCTGCTGATTTGG-30 and 50 -TTACATTGCAGATTGGAAAATCATCTCAGCGAGT-30 from A. thaliana cDNA. The PCR product was inserted into blunt-ended NcoI and HindIII sites of pFvG as described previously (Maehara et al. 2010b). The inserted nucleotide sequence for XI was confirmed using an ABI PRISM 310 genetic analyser (Applied Biosystems, Foster City, CA, USA).
Construction of XI transformants Flammulina velutipes Fv-1 strain was transformed with pFvGX as described previously (Maehara et al. 2010a). Transformants were selected on MYGS medium (0.4% malt extract, 0.4% yeast extract, 1% glucose and 0.5 M sucrose) containing 30 mg ml1 hygromycin B, and expression of the XI gene was confirmed by RT-PCR. cDNAs from each transformant were prepared as described above. Total DNA from each transformant was prepared using DNeasy Plant Maxi Kit (QIAGEN, CA, USA), and plasmid integration was confirmed by Southern hybridisation (Sambrook & Russell 2001), using the digoxigenin-labelled XI gene from Arabidopsis thaliana as a probe.
XI activity assays Flammulina velutipes mycelia were cultivated for 6 d and collected by centrifugation at 3000 g for 10 min, washed twice with 0.85% NaCl, collected by filtering through Miracloth (Calbiochem, California, USA) and squeezed to remove the water. The mycelia were frozen in liquid nitrogen and ground to fine powder. The fine powder was suspended in 100 mM TriseHCl (pH 7.5) and the suspension was centrifuged at 12000 g for 10 min. The supernatant was used as the cell extract. XI activity in the cell extract was determined at 30 C according to previously described methods (Kersters-Hilderson et al. 1987;
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Ethanol fermentation
Kuyper et al. 2003). The cell extract was added to a reaction mixture in a total volume of 1 ml, containing 100 mM TriseHCl buffer (pH 7.5), 10 mM MgCl2, 0.15 mM NADH, 500 mM xylose and 2 U sorbitol dehydrogenase (Wako, Osaka, Japan). The reaction was initiated by adding xylose. The amount of NADH was measured spectrophotometrically at A340 nm (extinction coefficient ¼ 6220 M1$cm1). The XI activity of the transformants was normalised against the wild-type strain as a background control. One unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of substrate per minute. The assay was performed in triplicate.
Ethanol fermentation was performed as described previously (Mizuno et al. 2009a) with some modifications. The Flammulina velutipes Fv-1 strain was cultivated aerobically in B medium with 120-rpm agitation at 25 C for 6 d. The mycelia were collected by centrifugation and washed with sterile water. The washed mycelia were mixed with fermentation buffer [1% yeast extract, 1% peptone, 0.1% KH2PO4 and 0.01% MgSO4$7H2O (pH 5.5)] and 1% w/v sugar in 15-ml falcon tubes and incubated semi-anaerobically at 25 C.
Ethanol and sugar determinations
Results
Ethanol concentrations were determined using an enzymatic test kit (Ethanol UV method (Cat.No.10176290035); Roche Diagnostics, Darmstadt, Germany). Concentrations of D-glucose, D-xylose and D-xylitol were determined using an HPLC system with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA).
Comparison of amino acid sequences of Arabidopsis thaliana XI with related sequences Fig 1 shows the alignment of the putative AtXI (Kaneko et al. 1998) with known xylose isomerases from Piromyces sp. strain
Fig 2 e Comparison of amino acid sequences of xylose isomerase from A. thaliana, Piromyces, S. rubiginosus and T. thermophilus. Amino acid sequences were aligned using ClustalW software (Thompson et al. 1997). Identical amino acids are shown in black boxes and similar amino acids are shown in grey boxes. B, Substrate binding residue; C, catalytic residue; O, metal binding residue.
Heterologous gene expression in Flammulina velutipes for ethanol production
E2 (accession number, CAB76571) (Harhangi et al. 2003), Streptomyces rubiginosus (accession number, AAA26838) (Carrell et al. 1989; Wong et al. 1991) and Thermus thermophilus (accession number, BAA14301) (Dekker et al. 1991). AtXI has a putative signal sequence at the N-terminus consisting of 22 amino acids. Comparison of amino acid sequence of mature region of AtXI with that of Piromyces XI revealed that AtXI showed moderate similarities to Piromyces XI with 52% identity. AtXI had only slight similarity with bacterial (S. rubiginosus and T. thermophilus) XI sequences (20% overall identity). The catalytic residue, metal binding residues and substrate binding residues that were identified in bacterial XIs were completely conserved in AtXI (Fig 2), suggesting that AtXI had XI activity.
Construction of XI transformants of Flammulina velutipes The pFvGX plasmid carried the mature region of AtXI between the gpd promoter and terminator to regulate expression of the AtXI gene and the hygromycin B phosphotransferase gene (hph) as a selection marker (Fig 1). After transformation of Fv-1 with pFvGX, approximately 100 colonies were obtained in selection plates. RT-PCR was performed to evaluate expression of the AtXI gene. mRNA expression of the heterologous XI gene in Flammulina velutipes was observed in only two transformants [Fv(AtXI )-1 and Fv(AtXI )-2] (Fig 3A, lanes 2 and 3). Using the digoxigenin-labelled AtXI gene as a probe, Southern blotting was performed to confirm whether pFvGX was integrated into the genomic DNA of these transformants (Fig 3B). No signal was detected in the genomic DNA of wild-type Fv1 (Fig 3B, lane WT), but significant hybridisation signals were detected in both transformants, with multiple signals in Fv(AtXI )-1 (Fig 3B, lane 2). These results indicated that at least a single AtXI gene was introduced into both transformants and appeared to be present in multiple copies in the genomic DNA of Fv(AtXI )-1 (Fig 3B, lane 2). Next, to determine whether AtXI was functionally expressed, XI activity of Fv(AtXI ) transformants was measured by monitoring NADH. As the result, apparent XI
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activity was detected in the Fv(AtXI ) transformants (data not shown). XI activity in Fv(AtXI )-2 was 2.5-fold higher than that in Fv(AtXI )-1. When whole-cell extracts were electrophoresed on SDSePAGE, no significant difference were observed between the wild-type Fv-1 and the transformants (data not shown), even though the transformant cell extracts had evident XI activity. Thus, it could be confirmed that the putative XI sequence from Arabidopsis thaliana was functionally expressed in Flammulina velutipes as XI, and ethanol production ability of both transformants was examined.
Production of ethanol from D-xylose by XI transformants of Flammulina velutipes To investigate whether the introduced AtXI improved the production of ethanol from D-xylose, ethanol fermentation by the transformants was examined. As shown in Fig 4A, the pattern of ethanol production and sugar consumption by the wildtype Fv-1 strain and Fv(AtXI ) transformants were similar when D-glucose was used as the carbon source. Almost all of the D-glucose was consumed within 2 d in all cases, and the wild-type and Fv(AtXI ) strains produced approximately 4 g L1 ethanol from 1% (w/v) D-glucose. The maximum conversion rate was approximately 80% of the theoretical conversion rate from D-glucose (Fig 4A). These results indicated that the introduction of the AtXI gene did not affect D-glucose metabolism to ethanol. In contrast, a marked difference in ethanol production from D-xylose was observed between the wild-type Fv-1 strain and Fv(AtXI ) transformants. Up to 0.7 and 1 g L1 ethanol was produced from 1% (w/v) D-xylose by Fv(AtXI )-1 and Fv(AtXI )-2, respectively, and the maximum conversion rates were approximately 16% and 25%, respectively. These rates were approximately 3- to 5-fold higher than that of the wild-type strain (Fig 4B). D-Xylose uptake by the Fv(AtXI ) transformants was slightly faster than that of the wild-type strain, although much lower than D-glucose uptake (Fig 4C). Significant xylitol accumulation was not observed in either the wild-type or
Fig 3 e Analysis of F. velutipes transformants. (A) RT-PCR-based analysis of xylose isomerase gene expression. Upper panel, AtXI gene; lower panel, b-tubulin gene as a control. Lane M, DNA molecular size markers (values on left); lane WT, wild-type Fv-1 as a negative control; lanes 1, 2 and 3, AtXI transformants. (B) Southern blotting of SpeI-digested genomic DNA using an hph gene probe. 5 mg of genomic DNA from transformants was digested with SpeI. Lane WT, wild-type Fv-1 as a negative control; lane M, DNA molecular size markers (values on left); lane 1, pFvGX (7828 bp) plasmid as a positive control; lanes 2 and 3, genomic DNA from recombinant strains Fv(AtXI )-1 and Fv(AtXI )-2, respectively.
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Fig 4 e Ethanol production by F. velutipes wild-type and Fv(AtXI ) transformant strains. (A) Ethanol production from 1% (w/v) D-glucose. Closed symbols, ethanol; open symbols, D-glucose. Circles, wild-type Fv-1 strain; squares, Fv(AtXI )-1 transformant; triangles, Fv(AtXI )-2 transformant. (B) Ethanol production from 1% (w/v) D-xylose. Closed circles, wildtype Fv-1; squares, Fv(AtXI )-1 transformant; triangles, Fv(AtXI )-2 transformant. (C) D-Xylose and xylitol concentrations in the ethanol fermentation medium. Open symbols, D-xylose; closed symbols, xylitol. Circles, wild-type Fv1 strain; squares, Fv(AtXI )-1 transformant; triangles, Fv(AtXI )-2 transformant. Error bars indicate standard deviations (n [ 3). Fv(AtXI )-1, however, xylitol accumulation was slightly increased in Fv(AtXI )-2. These results suggested that AtXI was functional in the pentose metabolism pathway of Flammulina velutipes.
Discussion In our previous studies, we screened the wild-type strain Fv-1 of Flammulina velutipes as an ethanol-producing organism for CBP. We evaluated its ethanol-producing ability and developed a method for gene transformation in this strain.
T. Maehara et al.
XIs have been used to improve the ethanol yield from D-xylose in Saccharomyces cerevisiae, which is a major ethanol producer. In general, XI is involved in the D-xylose metabolism in bacteria, but is not present in eukaryotes. Although there are some exceptions, the prokaryotic XI gene has been difficult to express in S. cerevisiae (Sarthy et al. 1987; Amore et al. 1989; Moes et al. 1996; Walfridsson et al. 1996; Brat et al. 2009), and desirable results have not been obtained. To overcome this problem, XI genes from anaerobic fungi (e.g. Piromyces sp. strain E2 and Orpinomyces) have been examined and successfully expressed in S. cerevisiae (Kuyper et al. 2003; Madhavan et al. 2009). Recent developments in genome biology revealed that plants may have a pentose-metabolising pathway that utilises XI. However, the study of pentose metabolism in plants is limited and only XI from barley has been reported (Kristo et al. 1996). Using this information, we attempted to use a putative XI gene from Arabidopsis thaliana for the transformation of F. velutipes. Our results clearly indicated that F. velutipes can functionally express the Arabidopsis thaliana XI gene, suggesting the possibility of using basidiomycetes as useful hosts for heterologous gene expression and the possibility of expanding the sources of genes for the transformation of F. velutipes to improve their characteristics. Flammulina velutipes mainly observed on hardwood in nature, and it has been thought to be consumed wood components (cellulose and hemicellulose) for their growth. While F. velutipes consumed glucose preferentially than xylose in xyloseeglucose medium in vitro, it can utilize xylose and xylan for its growth under aerobic conditions (data not shown). Furthermore, no metabolite products derived from xylose did not observed when sugar alcohols and free sugars in the fruit body of F. velutipes are measured. These observations suggest that F. velutipes metabolize xylose (Kitamoto & Gruen 1976; Takabatake et al. 2003) and it has a pentose metabolic pathway along with hexose metabolic pathway. However, under anaerobic conditions as an ethanol fermentation, F. velutipes is able to produce ethanol from glucose but not able to metabolize pentose effectively (Fig 4). Transformants containing AtXI showed increased ethanol production from D-xylose, but the conversion rate was not very high and there is scope for further improvement. In Fv(AtXI ), it is possible that ethanol production was increased by improved D-xylose uptake resulting from AtXI-catalysed conversion of D-xylose to D-xylulose. Because xylitol accumulation was observed in Fv(AtXI )-2, which had higher XI activity, it would be expected that the imbalance in the endogenous D-xylose pathway was caused by the increased conversion of D-xylose to D-xylulose through the action of AtXI or by the conversion of D-xylulose produced by AtXI to D-xylitol through the action of endogenous XDH. In general, D-xylulose convert to xylulose 5-phosphate by xylulokinase (XK). But it has been known that the endogenous XK activity is low or undetectable in Saccharomyces, and in F. velutipes, XK activity was not detected (data not shown). Probably, XK activity of F. velutipes is also low, a part of D-xylulose which is not converted ethanol would be changed into D-xylitol. These imbalances in the D-xylose pathway may be solved by expressing XK and regulation of the amounts of oxygen as demonstrated in S. cerevisiae (Eliasson et al. 2000; Toivari
Heterologous gene expression in Flammulina velutipes for ethanol production
et al. 2001). Knockout of endogenous XR and XDH in F. velutipes or expression of XR and XDH that are superior to the endogenous enzymes may also be effective approaches for improving ethanol production from D-xylose. The expression level of AtXI in F. velutipes appeared to be relatively low because a clear band corresponding to AtXI was not observed on SDSePAGE and the intensity of AtXI band was lower than that of b-tubulin (Fig 3A). AtXI may be digested by endogenous proteinases in F. velutipes, because it is known that basidiomycete fungi possess many proteases et al. 2007; Erjavec et al. 2012). In various organisms, (Sabotic protease-deficient strains have been used for heterologous gene expression (Buell et al. 1985; Brodin et al. 1986; Meerman & Georgiou 1994; Fahnestock & Fisher 1987; Honjo et al. 1987; Sander et al. 1994; Maehara et al. 2008). It may be possible that native protease(s) produced by the host cells is responsible for degradation of the products. However, proteases that can affect heterologous gene expression in F. velutipes are poorly understood. In conclusion, improvement of D-xylose fermentation by F. velutipes was demonstrated by transformation with a gene encoding XI from A. thaliana. The present study is the first report of an improved ability in F. velutipes through genetic engineering. Thus, we demonstrate the possibility of producing a useful strain of F. velutipes for CBP.
Acknowledgements This work was financially supported by a Grant-in-Aid (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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journal homepage: www.elsevier.com/locate/funbio
Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes Tomoko MAEHARAa, Koji TAKABATAKEb, Satoshi KANEKOa,* a
Food Biotechnology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan b Forest Institute, Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, 3 Yoshimine, Tateyama, Toyama 930-1362, Japan
article info
abstract
Article history:
To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isom-
Received 18 March 2013
erase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the
Received in revised form
gene expression was evaluated in transformants. mRNA expression of the putative XI gene
4 September 2013
and XI activity were observed in two transformants, indicating that the putative gene from
Accepted 30 September 2013
A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, eth-
Available online 12 October 2013
anol production from xylose was increased in the recombinant strains. This is the first re-
Corresponding Editor:
port demonstrating the possibility of using plant genes as candidates for improving the
Daniel Eastwood
characteristics of F. velutipes. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Basidiomycete fungi Ethanol fermentation Flammulina velutipes Heterologous gene expression Xylose metabolism
Introduction To achieve an economic bioethanol production from lignocellulosic biomass, consolidated bioprocessing (CBP) that combines enzyme production, enzymatic saccharification and ethanol fermentation in one step was proposed by Lynd et al. (2005). Recently, several studies of CBP capable of high yield conversion of biomass to ethanol were reported using the combined abilities of two organisms such as a cellulolytic enzyme-producing organism and an ethanol-producing organism (Zhang & Zhang 2010; van Zyl et al. 2011). However, no single organism suitable for CBP has been developed.
Flammulina velutipes is a wood-rotting fungus that can completely degrade lignocellulose. Because the F. velutipes Fv-1 strain produces both ethanol and cellulolytic enzymes, we investigated CBP bioethanol production using F. velutipes and found that this strain was a good candidate for ethanol production from lignocelluloses by CBP (Mizuno et al. 2009a, 2009b; Maehara et al. 2013). Furthermore, we have developed gene transformation methods for F. velutipes (Maehara et al. 2010a, 2010b). The Fv-1 strain was able to ferment D-glucose, D-fructose, D-mannose, sucrose, maltose, cellobiose and cellulose; however, it can hardly produce ethanol from pentoses (Maehara et al. 2013). Because lignocellulosic biomass contains
* Corresponding author. Tel.: þ81 29 838 8063; fax: þ81 29 838 7996. E-mail address: [email protected] (S. Kaneko). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.09.005
Heterologous gene expression in Flammulina velutipes for ethanol production
up to approximately 30e40% pentoses, utilisation of pentoses is necessary for effective ethanol production. Therefore, improvement of pentose fermentation by F. velutipes would be required. A major bioethanol producer, Saccharomyces cerevisiae is not able to ferment pentoses. Thus, many studies have been conducted for the genetic engineering of this yeast directed at improving pentose fermentation (Jeffries & Jin 2004; Matsushika et al. 2009). There have been two general approaches to this work. The first approach has been the expression of heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes to improve the endogenous pentose-metabolising pathway. Recombinant S. cerevisiae strains that expressed XR and XDH showed improved ethanol production from D-xylose. However, xylitol accumulation caused by an NADPH/NAD cofactor imbalance under anaerobic condition was observed; thus, it would be desirable to introduce additional enzyme-encoding genes to address this problem. The second approach has been the expression of a heterologous xylose isomerase (XI) gene to produce a new bypass pathway for pentose metabolism to convert D-xylose to D-xylulose directly. Because XI does not require redox cofactors, it does not cause xylitol accumulation and cofactor imbalances; however, in general, XI is involved in the pentose pathway of bacteria and is difficult to express in eukaryotes. Because F. velutipes is expected to have the same sugar metabolism pathway as S. cerevisiae (Barnett 1976; Maehara et al. 2013) and because the introduction of multiple enzymeencoding genes into F. velutipes would be difficult, we aimed to introduce a heterologous XI gene from a eukaryotic organism into F. velutipes. In this study, we focused on the gene encoding a putative XI from the plant Arabidopsis thaliana (AtXI ). This putative gene was transformed into the F. velutipes Fv-1 strain, and its expression and effect on ethanol production from D-xylose were evaluated in F. velutipes. To our knowledge, there is no report on the use of a plant XI gene for improving pentose fermentation and our study is the first report on improving the characteristics of F. velutipes by genetic engineering.
Materials and methods Strains, media and culture conditions The wood-rotting fungus Flammulina velutipes Fv-1 strain was used as the transformation host. The strain was grown at 25 C with shaking at 120 rpm in B medium [1% yeast extract, 1% peptone, 1% D-glucose, 0.1% KH2PO4 and 0.01% MgSO4$7H2O (pH 5.5)]. Hygromycin B (30 mg ml1) was added to the cultivation medium if required. The bacterial strain Escherichia coli DH5a was used for cloning AtXI. The E. coli strain was grown at 37 C in LuriaeBertani (LB) medium. Ampicillin (100 mg ml1) was added to the medium when required. The seeds of Arabidopsis thaliana were cultivated at 20 C on MurashigeeSkoog medium supplemented with 2% sucrose, vitamin solution (0.3% thiamine HCl, 0.5% nicotinic acid and 0.05% pyridoxine HCl) and 0.3% agar.
Plasmid construction The plasmid used in this study is shown in Fig 1. The plasmid designated pFvGX carried a 1368-bp XI gene fragment isolated
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Fig 1 e Structures of the plasmid used in this study. pFvGX (7828 bp) was derived from pFvG (Maehara et al. 2010a) by cloning the AtXI gene without a signal sequence into the multi-cloning sites (MCS) of pFvG. hph is a hygromycin B phosphotransferase gene and ampr is an ampicillin resistance gene. gpd is glyceraldehydes-3-phosphate dehydrogenase and trp1 is the tryptophan synthetase gene.
from Arabidopsis thaliana. Total RNA of A. thaliana was prepared using RNeasy Plant Mini Kit (QIAGEN, CA, USA), and cDNA was prepared by reverse transcriptase (RT) reactions using ReverTra Ace-a (Toyobo, Osaka, Japan) with 0.1 ng of total RNA as the template. The gene encoding XI from A. thaliana was amplified by PCR with KOD-plus- ver.2 (Toyobo, Osaka, Japan) using the primer pairs 50 -GATCCACCAACATGTCCTGCTGATTTGG-30 and 50 -TTACATTGCAGATTGGAAAATCATCTCAGCGAGT-30 from A. thaliana cDNA. The PCR product was inserted into blunt-ended NcoI and HindIII sites of pFvG as described previously (Maehara et al. 2010b). The inserted nucleotide sequence for XI was confirmed using an ABI PRISM 310 genetic analyser (Applied Biosystems, Foster City, CA, USA).
Construction of XI transformants Flammulina velutipes Fv-1 strain was transformed with pFvGX as described previously (Maehara et al. 2010a). Transformants were selected on MYGS medium (0.4% malt extract, 0.4% yeast extract, 1% glucose and 0.5 M sucrose) containing 30 mg ml1 hygromycin B, and expression of the XI gene was confirmed by RT-PCR. cDNAs from each transformant were prepared as described above. Total DNA from each transformant was prepared using DNeasy Plant Maxi Kit (QIAGEN, CA, USA), and plasmid integration was confirmed by Southern hybridisation (Sambrook & Russell 2001), using the digoxigenin-labelled XI gene from Arabidopsis thaliana as a probe.
XI activity assays Flammulina velutipes mycelia were cultivated for 6 d and collected by centrifugation at 3000 g for 10 min, washed twice with 0.85% NaCl, collected by filtering through Miracloth (Calbiochem, California, USA) and squeezed to remove the water. The mycelia were frozen in liquid nitrogen and ground to fine powder. The fine powder was suspended in 100 mM TriseHCl (pH 7.5) and the suspension was centrifuged at 12000 g for 10 min. The supernatant was used as the cell extract. XI activity in the cell extract was determined at 30 C according to previously described methods (Kersters-Hilderson et al. 1987;
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Ethanol fermentation
Kuyper et al. 2003). The cell extract was added to a reaction mixture in a total volume of 1 ml, containing 100 mM TriseHCl buffer (pH 7.5), 10 mM MgCl2, 0.15 mM NADH, 500 mM xylose and 2 U sorbitol dehydrogenase (Wako, Osaka, Japan). The reaction was initiated by adding xylose. The amount of NADH was measured spectrophotometrically at A340 nm (extinction coefficient ¼ 6220 M1$cm1). The XI activity of the transformants was normalised against the wild-type strain as a background control. One unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of substrate per minute. The assay was performed in triplicate.
Ethanol fermentation was performed as described previously (Mizuno et al. 2009a) with some modifications. The Flammulina velutipes Fv-1 strain was cultivated aerobically in B medium with 120-rpm agitation at 25 C for 6 d. The mycelia were collected by centrifugation and washed with sterile water. The washed mycelia were mixed with fermentation buffer [1% yeast extract, 1% peptone, 0.1% KH2PO4 and 0.01% MgSO4$7H2O (pH 5.5)] and 1% w/v sugar in 15-ml falcon tubes and incubated semi-anaerobically at 25 C.
Ethanol and sugar determinations
Results
Ethanol concentrations were determined using an enzymatic test kit (Ethanol UV method (Cat.No.10176290035); Roche Diagnostics, Darmstadt, Germany). Concentrations of D-glucose, D-xylose and D-xylitol were determined using an HPLC system with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA).
Comparison of amino acid sequences of Arabidopsis thaliana XI with related sequences Fig 1 shows the alignment of the putative AtXI (Kaneko et al. 1998) with known xylose isomerases from Piromyces sp. strain
Fig 2 e Comparison of amino acid sequences of xylose isomerase from A. thaliana, Piromyces, S. rubiginosus and T. thermophilus. Amino acid sequences were aligned using ClustalW software (Thompson et al. 1997). Identical amino acids are shown in black boxes and similar amino acids are shown in grey boxes. B, Substrate binding residue; C, catalytic residue; O, metal binding residue.
Heterologous gene expression in Flammulina velutipes for ethanol production
E2 (accession number, CAB76571) (Harhangi et al. 2003), Streptomyces rubiginosus (accession number, AAA26838) (Carrell et al. 1989; Wong et al. 1991) and Thermus thermophilus (accession number, BAA14301) (Dekker et al. 1991). AtXI has a putative signal sequence at the N-terminus consisting of 22 amino acids. Comparison of amino acid sequence of mature region of AtXI with that of Piromyces XI revealed that AtXI showed moderate similarities to Piromyces XI with 52% identity. AtXI had only slight similarity with bacterial (S. rubiginosus and T. thermophilus) XI sequences (20% overall identity). The catalytic residue, metal binding residues and substrate binding residues that were identified in bacterial XIs were completely conserved in AtXI (Fig 2), suggesting that AtXI had XI activity.
Construction of XI transformants of Flammulina velutipes The pFvGX plasmid carried the mature region of AtXI between the gpd promoter and terminator to regulate expression of the AtXI gene and the hygromycin B phosphotransferase gene (hph) as a selection marker (Fig 1). After transformation of Fv-1 with pFvGX, approximately 100 colonies were obtained in selection plates. RT-PCR was performed to evaluate expression of the AtXI gene. mRNA expression of the heterologous XI gene in Flammulina velutipes was observed in only two transformants [Fv(AtXI )-1 and Fv(AtXI )-2] (Fig 3A, lanes 2 and 3). Using the digoxigenin-labelled AtXI gene as a probe, Southern blotting was performed to confirm whether pFvGX was integrated into the genomic DNA of these transformants (Fig 3B). No signal was detected in the genomic DNA of wild-type Fv1 (Fig 3B, lane WT), but significant hybridisation signals were detected in both transformants, with multiple signals in Fv(AtXI )-1 (Fig 3B, lane 2). These results indicated that at least a single AtXI gene was introduced into both transformants and appeared to be present in multiple copies in the genomic DNA of Fv(AtXI )-1 (Fig 3B, lane 2). Next, to determine whether AtXI was functionally expressed, XI activity of Fv(AtXI ) transformants was measured by monitoring NADH. As the result, apparent XI
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activity was detected in the Fv(AtXI ) transformants (data not shown). XI activity in Fv(AtXI )-2 was 2.5-fold higher than that in Fv(AtXI )-1. When whole-cell extracts were electrophoresed on SDSePAGE, no significant difference were observed between the wild-type Fv-1 and the transformants (data not shown), even though the transformant cell extracts had evident XI activity. Thus, it could be confirmed that the putative XI sequence from Arabidopsis thaliana was functionally expressed in Flammulina velutipes as XI, and ethanol production ability of both transformants was examined.
Production of ethanol from D-xylose by XI transformants of Flammulina velutipes To investigate whether the introduced AtXI improved the production of ethanol from D-xylose, ethanol fermentation by the transformants was examined. As shown in Fig 4A, the pattern of ethanol production and sugar consumption by the wildtype Fv-1 strain and Fv(AtXI ) transformants were similar when D-glucose was used as the carbon source. Almost all of the D-glucose was consumed within 2 d in all cases, and the wild-type and Fv(AtXI ) strains produced approximately 4 g L1 ethanol from 1% (w/v) D-glucose. The maximum conversion rate was approximately 80% of the theoretical conversion rate from D-glucose (Fig 4A). These results indicated that the introduction of the AtXI gene did not affect D-glucose metabolism to ethanol. In contrast, a marked difference in ethanol production from D-xylose was observed between the wild-type Fv-1 strain and Fv(AtXI ) transformants. Up to 0.7 and 1 g L1 ethanol was produced from 1% (w/v) D-xylose by Fv(AtXI )-1 and Fv(AtXI )-2, respectively, and the maximum conversion rates were approximately 16% and 25%, respectively. These rates were approximately 3- to 5-fold higher than that of the wild-type strain (Fig 4B). D-Xylose uptake by the Fv(AtXI ) transformants was slightly faster than that of the wild-type strain, although much lower than D-glucose uptake (Fig 4C). Significant xylitol accumulation was not observed in either the wild-type or
Fig 3 e Analysis of F. velutipes transformants. (A) RT-PCR-based analysis of xylose isomerase gene expression. Upper panel, AtXI gene; lower panel, b-tubulin gene as a control. Lane M, DNA molecular size markers (values on left); lane WT, wild-type Fv-1 as a negative control; lanes 1, 2 and 3, AtXI transformants. (B) Southern blotting of SpeI-digested genomic DNA using an hph gene probe. 5 mg of genomic DNA from transformants was digested with SpeI. Lane WT, wild-type Fv-1 as a negative control; lane M, DNA molecular size markers (values on left); lane 1, pFvGX (7828 bp) plasmid as a positive control; lanes 2 and 3, genomic DNA from recombinant strains Fv(AtXI )-1 and Fv(AtXI )-2, respectively.
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Fig 4 e Ethanol production by F. velutipes wild-type and Fv(AtXI ) transformant strains. (A) Ethanol production from 1% (w/v) D-glucose. Closed symbols, ethanol; open symbols, D-glucose. Circles, wild-type Fv-1 strain; squares, Fv(AtXI )-1 transformant; triangles, Fv(AtXI )-2 transformant. (B) Ethanol production from 1% (w/v) D-xylose. Closed circles, wildtype Fv-1; squares, Fv(AtXI )-1 transformant; triangles, Fv(AtXI )-2 transformant. (C) D-Xylose and xylitol concentrations in the ethanol fermentation medium. Open symbols, D-xylose; closed symbols, xylitol. Circles, wild-type Fv1 strain; squares, Fv(AtXI )-1 transformant; triangles, Fv(AtXI )-2 transformant. Error bars indicate standard deviations (n [ 3). Fv(AtXI )-1, however, xylitol accumulation was slightly increased in Fv(AtXI )-2. These results suggested that AtXI was functional in the pentose metabolism pathway of Flammulina velutipes.
Discussion In our previous studies, we screened the wild-type strain Fv-1 of Flammulina velutipes as an ethanol-producing organism for CBP. We evaluated its ethanol-producing ability and developed a method for gene transformation in this strain.
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XIs have been used to improve the ethanol yield from D-xylose in Saccharomyces cerevisiae, which is a major ethanol producer. In general, XI is involved in the D-xylose metabolism in bacteria, but is not present in eukaryotes. Although there are some exceptions, the prokaryotic XI gene has been difficult to express in S. cerevisiae (Sarthy et al. 1987; Amore et al. 1989; Moes et al. 1996; Walfridsson et al. 1996; Brat et al. 2009), and desirable results have not been obtained. To overcome this problem, XI genes from anaerobic fungi (e.g. Piromyces sp. strain E2 and Orpinomyces) have been examined and successfully expressed in S. cerevisiae (Kuyper et al. 2003; Madhavan et al. 2009). Recent developments in genome biology revealed that plants may have a pentose-metabolising pathway that utilises XI. However, the study of pentose metabolism in plants is limited and only XI from barley has been reported (Kristo et al. 1996). Using this information, we attempted to use a putative XI gene from Arabidopsis thaliana for the transformation of F. velutipes. Our results clearly indicated that F. velutipes can functionally express the Arabidopsis thaliana XI gene, suggesting the possibility of using basidiomycetes as useful hosts for heterologous gene expression and the possibility of expanding the sources of genes for the transformation of F. velutipes to improve their characteristics. Flammulina velutipes mainly observed on hardwood in nature, and it has been thought to be consumed wood components (cellulose and hemicellulose) for their growth. While F. velutipes consumed glucose preferentially than xylose in xyloseeglucose medium in vitro, it can utilize xylose and xylan for its growth under aerobic conditions (data not shown). Furthermore, no metabolite products derived from xylose did not observed when sugar alcohols and free sugars in the fruit body of F. velutipes are measured. These observations suggest that F. velutipes metabolize xylose (Kitamoto & Gruen 1976; Takabatake et al. 2003) and it has a pentose metabolic pathway along with hexose metabolic pathway. However, under anaerobic conditions as an ethanol fermentation, F. velutipes is able to produce ethanol from glucose but not able to metabolize pentose effectively (Fig 4). Transformants containing AtXI showed increased ethanol production from D-xylose, but the conversion rate was not very high and there is scope for further improvement. In Fv(AtXI ), it is possible that ethanol production was increased by improved D-xylose uptake resulting from AtXI-catalysed conversion of D-xylose to D-xylulose. Because xylitol accumulation was observed in Fv(AtXI )-2, which had higher XI activity, it would be expected that the imbalance in the endogenous D-xylose pathway was caused by the increased conversion of D-xylose to D-xylulose through the action of AtXI or by the conversion of D-xylulose produced by AtXI to D-xylitol through the action of endogenous XDH. In general, D-xylulose convert to xylulose 5-phosphate by xylulokinase (XK). But it has been known that the endogenous XK activity is low or undetectable in Saccharomyces, and in F. velutipes, XK activity was not detected (data not shown). Probably, XK activity of F. velutipes is also low, a part of D-xylulose which is not converted ethanol would be changed into D-xylitol. These imbalances in the D-xylose pathway may be solved by expressing XK and regulation of the amounts of oxygen as demonstrated in S. cerevisiae (Eliasson et al. 2000; Toivari
Heterologous gene expression in Flammulina velutipes for ethanol production
et al. 2001). Knockout of endogenous XR and XDH in F. velutipes or expression of XR and XDH that are superior to the endogenous enzymes may also be effective approaches for improving ethanol production from D-xylose. The expression level of AtXI in F. velutipes appeared to be relatively low because a clear band corresponding to AtXI was not observed on SDSePAGE and the intensity of AtXI band was lower than that of b-tubulin (Fig 3A). AtXI may be digested by endogenous proteinases in F. velutipes, because it is known that basidiomycete fungi possess many proteases et al. 2007; Erjavec et al. 2012). In various organisms, (Sabotic protease-deficient strains have been used for heterologous gene expression (Buell et al. 1985; Brodin et al. 1986; Meerman & Georgiou 1994; Fahnestock & Fisher 1987; Honjo et al. 1987; Sander et al. 1994; Maehara et al. 2008). It may be possible that native protease(s) produced by the host cells is responsible for degradation of the products. However, proteases that can affect heterologous gene expression in F. velutipes are poorly understood. In conclusion, improvement of D-xylose fermentation by F. velutipes was demonstrated by transformation with a gene encoding XI from A. thaliana. The present study is the first report of an improved ability in F. velutipes through genetic engineering. Thus, we demonstrate the possibility of producing a useful strain of F. velutipes for CBP.
Acknowledgements This work was financially supported by a Grant-in-Aid (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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