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Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization Chengqiang Wang, Xiaoming Bao, Yanwei Li, Chunlei Jiao, Jin Hou, Qingzhu Zhang, Weixin Zhang, Weifeng Liu, Yu Shen www.elsevier.com/locate/ymben

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S1096-7176(15)00060-9 http://dx.doi.org/10.1016/j.ymben.2015.04.007 YMBEN997

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Metabolic Engineering

Received date: 22 January 2015 Revised date: 25 March 2015 Accepted date: 24 April 2015 Cite this article as: Chengqiang Wang, Xiaoming Bao, Yanwei Li, Chunlei Jiao, Jin Hou, Qingzhu Zhang, Weixin Zhang, Weifeng Liu, Yu Shen, Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2015.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization

Chengqiang Wang 1,a,b, Xiaoming Bao1,b, Yanwei Li2, Chunlei Jiao1 , Jin Hou1, Qingzhu Zhang2, Weixin Zhang1, Weifeng Liu1 , Yu Shen1,*

1

The State Key Laboratory of Microbial Technology, Shandong University, Shan

Da Nan Road 27, Jinan, 250100, P. R. China 2

Environment Research Institute, Shandong University, Shan Da Nan Road 27,

Jinan, 250100, P. R. China

a

Present address: College of Life Sciences, Shandong Agricultural

University/Shandong Key Laboratory of Agricultural Microbiology, Daizong Street 61, Taian, 271018, P. R. China. b

These authors contributed equally to this paper.

* Corresponding author

Mailing address: The State Key Laboratory of Microbial Technology, Shandong University, Shan Da Nan Road 27, Jinan, 250100, China. Phone and Fax: +86 1

531 88365826. E-mail address: [email protected].

Abstract Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae. Eight heterologous sugar transporters were characterized in S.cerevisiae. The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332 to 336) in the seventh trans-membrane span plays an important role in D-xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent D-xylose from escaping during uptake. F432A and N360S mutations enhanced the D-xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported D-xylose without any glucose-inhibition, high lighting its potential application in constructing glucose-xylose co-fermentation strains for biomass refining.

Keywords: transporter; D-xylose; pentose; budding yeast; heterologous; protein engineering

1. Introduction Bioethanol, which is produced from lignocellulosic materials, is an attractive 2

alternative source of fossil fuels (Hahn-Hägerdal et al., 2006). In addition to the major component glucose, the lignocellulosic materials are composed of approximately 30% pentoses, mainly D-xylose and L-arabinose (Seiboth and Metz, 2011; Subtil and Boles, 2011). Cofermenting the pentoses and glucose synchronously is essential for cost-effective bioethanol production (Kim et al., 2012). Saccharomyces cerevisiae is a well-studied microorganism that is widely used in traditional ethanol production, with excellent features of safety, robustness, high tolerance to inhibitors, etc. (Du et al., 2010; Jeffries, 2006). Aiming to endow the S. cerevisiae with the capacity of fermenting D-xylose and L-arabinose, the initial utilization pathways of the two pentoses were introduced into the strains (Amore et al., 1991; Bera et al., 2010; Eliasson et al., 2000; Jin et al., 2004; Karhumaa et al., 2006; Kim et al., 2013; Kötter et al., 1990; Walfridsson et al., 1995; Wang et al., 2013b); however, these engineered strains metabolize glucose, D-xylose, or L-arabinose sequentially, and their D-xylose and L-arabinose consumption rates were much lower than that of glucose.

Transmembrane transport is the first step in the sugar metabolic pathway; however, there is no specific D-xylose or L-arabinose transporter in S. cerevisiae. The D-xylose and L-arabinose uptake by S. cerevisiae depends upon hexose transporters and is competitively inhibited by glucose (Farwick et al., 2014). This finding was considered as one of the important reasons underlying the lower D-xylose and L-arabinose utilization compared to glucose. The discovery of more efficient and specific transporters is necessary to enhance the D-xylose and L-arabinose uptake, and therefore promote the metabolic capacity of S. cerevisiae. To study the function of sugar transporters without the background noise of native hexose transporters, the strains lacking all 18 of the native hexose transporters (hxt null) (Wieczorke et al., 3

1999) or eight main hexose transporters Hxt1-7p and Gal2p (Kruckeberg et al., 1999) have been used as powerful tools. The endogenous transporter Gal2p exhibited a relatively high transport capacity for both D-xylose and L-arabinose, although the transport activity was strongly inhibited by glucose (Leandro et al., 2009; Subtil and Boles, 2011; Young et al., 2011).

The native pentose-utilizing microorganisms are considered to be good sources of pentose transporters (Leandro et al., 2009; Young et al., 2010). Many heterologous pentose transporters have been expressed in S. cerevisiae, such as Gxf1p and Gxs1p from Candida intermedia; Sut1p, Sut4p, Xut1p, Xut3p, Xut4p, Xut5p, Xut6p, Xut7p, Xyp29p, Rgt2p, and AraTp from Scheffersomyces stipitis; Stp2p, At5g59250p, and At5g17010p from Arabidopsis thaliana; XylHPp, 2C02530p, and 2D01474p from Debaryomyces hansenii; An25p from Neurospora crassa, Lat1p and Lat2p from Ambrosiozyma monospora, etc. (Du et al., 2010; Hector et al., 2008; Katahira et al., 2008; Leandro et al., 2006; Ma et al., 2012; Moon et al., 2013; Runquist et al., 2009; Young et al., 2011; Young et al., 2014). Some of these transporters can increase intracellular D-xylose accumulation in the hxt null strain and (or) confer the growth of the hxt null strain containing the D-xylose initial metabolic pathway. AraTp and Stp2p functionally mediate the uptake of L-arabinose (Subtil and Boles, 2011). Lat1p and Lat2p were proven to be L-arabinose specific transporters (Verho et al., 2011); however, to date, the pentose uptake capacity using heterologous transporters is still low. The main challenge remains the contradiction between the specificity and efficiency of D-xylose transport.

The molecular modifications carried on heterologous or endogenous transporters altered their xylose transport performance, especially when glucose was present. The 4

mutant Gxs1p (gxs1-2.3) enhanced the growth rate of recombinant hxt null S. cerevisiae on D-xylose by 70% compared to wild-type Gxs1p (Young et al., 2012). Then, the motif G-G/F-XXX-G in the first transmembrane span was determined to be important for sugar transport preference (Young et al., 2014). Additionally, an S. cerevisiae endogenous transporter mutant Gal2p (N376F), which prominently increased D-xylose transport preference, was screened out (Farwick et al., 2014). The functional study results showed that glucose does not inhibit the transport of xylose by Gal2p (N376F) (Farwick et al., 2014); however, the xylose transport capacity of this mutant was 59 % lower than wild-type Gal2p (Farwick et al., 2014). Recently, a fusion protein Hxt36 deriving from endogenous transporters Hxt3p and Hxt6p was screened out in an evolved strain. Using the saturation mutagenesis strategy, the amino acid residue N367 in the TMS8 of this fusion transporter Hxt36 was demonstrated to be the key conserved residue for glucose inhibition free xylose transport (Nijland, et al., 2014). Until now, the mining or modification of the transporter(s) that can efficiently and specifically transport pentose without glucose inhibition during the co-fermentation of sugar mixtures has been required. Moreover, the mechanism of this process remains unclear.

In the present work, heterologous transporters were cloned from pentose native utilizers. Their characteristics were surveyed in hxt null S. cerevisiae and compared with native transporter Gal2p and the E.coli transporter XylE whose 3D-structure was solved. An efficient D-xylose transporter, Mgt05196p from Meyerozyma guilliermondii, was selected. The critical amino acids residues and motif for D-xylose transportation were predicted and then verified using site-directed mutagenesis to supplement the still limited structural understanding of glucose transporters. A mutant 5

with specific activity for D-xylose transportation and free from glucose inhibition was obtained, whichi has potential use in the biorefinery of lignocellulosic materials.

2. Materials and methods 2.1. Cloning of putative pentose transporters

The genomic DNA of two native pentose utilizing microorganisms, Trichosporon cutaneum (Hu et al., 2011) and Meyerozyma guilliermondii (Papon et al., 2013), were used to clone the putative pentose transporter genes. The primers designed according to the two conserved motifs PESPRXL and PETKGXXXE of sugar transporters (Leandro et al., 2009), were used to identify novel sugar transporters from the T. cutaneum genome due to the absence of sequence information. Then, two potential transporter ORFs, TCT1 and TCT2, were cloned from T. cutaneum AS 2.571 (Hu et al., 2011) using the SMART RACE cDNA Amplification Kit (Takara, Japan). Basing on the BLAST analysis, four potential transporters, Mgt04891p, Mgt05196p, Mgt05293p, and Mgt05860p, with>30% amino acid sequence identity to Gal2p were found in the entire genome of M. guilliermondii ATCC 6260 (GenBankaccession number AAFM01000000) (Papon et al., 2013).Then, the genes encoding these potential transporters were cloned from Candida guilliermondii (teleomorph M. guilliermondii) AS 2.1699 (China General Microbiological Culture Center, Beijing, China). Further, the genes STP1 from Trichoderma reeseiand AraE from Corynebacterium glutamicum ATCC31831 (Wang et al., 2013a; Zhang et al., 2013) were also selected as candidates.

2.2. Plasmid and strain construction

6

The plasmid pJFE3 containing the promoter TEF1p and terminator PGK1t with the selection marker gene URA3 (Shen et al., 2012) was used to express the genes of the transporters. The ORFs encoding the transporters and their mutants were inserted between the corresponding restriction enzyme sites between TEF1p and PGK1t in pJFE3. The fusion PCR strategy based on overlap extension polymerase chain reaction (Urban et al., 1997) was used in site-directed mutation to obtain the transporter mutants. The fragment of the MGT05196 gene without a stop codon was inserted into the plasmid pJFE3-yEGFP3 in front of the encoding sequence of yEGFP3 (Wang et al., 2013a), resulting in pJFE3-MGT05196-yEGFP3. All of the plasmids based on pJFE3 were transformed into the hxt null strain EBY.VW4000, which lacks all of the 18 native hexose transporters and, therefore, uses maltose as a culture carbon source (Wieczorke et al., 1999). Moreover, the basic D-xylose metabolic pathway was built into the hxt null background strains using the integrated plasmid pYMIK-xy127 as reported previously (Wang et al., 2004).

The plasmids and S. cerevisiae strains used in the present work are listed in Table 1. The primers used in the present work and the corresponding restriction enzyme sites used in plasmid construction are summarized in Supplementary Table 1.

2.3. Media and growth measurement YPM (10 g L-1 peptone, 20 g L-1 yeast extract, 20 g L-1 maltose) was used to culture EBY.VW4000 and derivative hxt null strains. SD medium containing 1.7 g L -1 yeast nitrogen base (YNB, Sangon, China) and 5 g L -1 ammonium sulfate (Sangon, China) supplemented with CSM-URA or CSM-LEU-URA (MP Biomedicals, Solon, 7

OH) and 20 g L-1 of one or two tested sugars were used for the culture of strains containing the plasmids according to the test requirements.

In the spotting assays, the cells were harvested from the 12 h culture in SD medium supplemented with CSM-URA and 20 g L-1 maltose, washed three times with ddH 2O, resuspended in 0.9% NaCl and incubated at 30 °C for 9 h to consume the endogenous substances. The density of the resuspended cells was normalized to an OD600 of 1.0. Then, 4 μL of the 10-fold serially diluted cells were dropped on the test plates and cultured at 30 °C. The growth status was measured after 5 days. The strains were cultured in the YPM or SD medium supplied 20 g L-1 maltose for 12 hours, and then transfer to the fresh medium with an initial OD600 of 0.1 and cultured for another 12 hours. This pre-cultured cells were collected and washed twice then used as seed cells for the tests of intracellular accumulation, symporter assay, and so on.

The strain biomass in the liquid was determined by measuring the culture optical density (OD600) using a BioPhotometer plus (Eppendorf, Germany). The exponential growth rates, which are the linear regression coefficients of ln OD600 versus time calculated from the growth curve, are used to compare the growth capacities (Peng et al., 2012; Young et al., 2014). The dry cell weight (DCW) was calculated according to the formula DCW (mg mL-1) = 0.2365×OD600+ 0.1149 (Wang et al., 2013a). The Bioscreen C system (Growth Curves, USA) was also used to monitor cell growth during the preliminary large-scale screening work (Young et al., 2014).

2.4. Intracellular accumulation of D-xylose or L-arabinose

The intracellular accumulation of D-xylose or L-arabinose was characterized in hxt null strain as previously reported (Du et al., 2010). The seed cells were transferred 8

into SD medium containing 20 g L-1 maltose and cultured for 10 h at 30 °C. Then, the cells were collected and resuspended in fresh SD medium supplied with 20 g L -1 D-xylose or 20 g L-1 L-arabinose. The final OD600 was 20. After an incubation time of 30, 60, and 120 min, 10 mL samples were collected. The cells were collected and washed with ice-cold ddH2O, then resuspended in ddH2O and incubated at 37 °C overnight to extract the intracellular sugars and sugar alcohols, the quantity of which was determined later using high performance liquid chromatography (HPLC). The total pentose and pentose alcohol per gram in the dried cells were used to characterize the accumulation amounts (Du et al., 2010). The parametric statistical method of Student’s t test was used in the significance analysis.

2.5. Symporter assay

To test if a D-xylose transporter was a symporter, the symporter assay (Du et al., 2010) was performed using an Orion 3-Star pH meter equipped with a real-time monitor (Thermo Scientific, USA, MA). First, 100mL of fresh culture (OD600 ~0.5) of the transporter-expressing hxt null cells were collected, washed three times with ice-cold ddH2O, and resuspended in 10 mL of ddH 2O-diluted HCl (pH 5).Then the pH value was recorded before and after the 200 μL of 40 g L-1 D-xylose was added. The increased value of the pH (shown as ΔpH) reflects the D-xylose/H + symport behavior of the D-xylose transporters.

2.6. Fluorescence microscopy A 10 h culture was harvested and resuspended in a 50 mmol l -1 PBS buffer (pH 6.5). The epifluorescence and phase-contrast images were taken using a Nikon ECLIPSE 80i system equipped with a plan Apochromat 40×objective (NA=0.95) 9

and a plan Apochromat 60× oil objective (NA=1.40),as previously reported (Chen et al., 2013).

2.7. Batch fermentation and products analysis

The cells were pre-cultured, collected, washed and then inoculated in 100-mL flasks with 40 mL of SD medium at 30 °C, 200 r min-1. The oxygen-limited and aerobic conditions were maintained using a rubber stopper and cotton plug on the shake flask, respectively.

The concentration of the sugars and metabolites were determined by HPLC using a Prominence LC-20A (Shimadzu, Japan) equipped with the refractive index detector RID-10A (Shimadzu, Japan). Glucose, D-xylose, D-xylitol and ethanol were analyzed using the Aminex HPX-87H ion exchange column (Bio-Rad, Hercules, USA) at 45°C, with a mobile phase of 5 mmol L-1 H2 SO4 at a flow rate of 0.6 mL min-1. L-arabinose and L-arabitol were analyzed using the Aminex HPX-87P ion exchange column (Bio-Rad, USA) at 80°C, with a mobile phase of water at a flow rate of 0.6 mL min-1 (Wang et al., 2013b).

2.8. Homologous modeling of Mgt05196p suggests crucial amino acid residues for D-xylose binding

The putative homology models of the transporter Mgt05196p and its critical amino acid residues D-xylose binding were analyzed using the software Discovery Studio (DS, Accelrys, USA, CA).The E.coli homologues of the glucose transporters GLUT1-4 (PDB code 4GBY, 4GBZ, and 4GC0) (Sun et al., 2012) served as the template to generate the homology model using the MODELER auto model in the 10

protein modeling module. The loop regions of the protein structure were refined using CHARMm-based molecular mechanics. The refined models were validated using PROCHECK (Laskowski et al., 1993). The location of D-xylose was determined using core-constrained protein docking and a modified CHARMm-based CDOCKER method. The best position among the determined ten positions of D-xylose was chosen by comparing the CDOCKER energies.

3. Results 3.1. Cloning and phylogenetic analysis of putative pentose transporters

Two natural D-xylose-utilizing strains, T. cutaneum, which can consume D-xylose and glucose simultaneously (Hu et al., 2011), and M. guilliermondii, which can effectively utilize D-xylose (Papon et al., 2013), were used for cloning the novel pentose transporters. By using the smart RACE, two putative sugar transporter genes were cloned out from T. cutaneum AS 2.571 and then their sequences were submitted to GenBank, which are TCT1 (accession number: KJ716333) and TCT2 (accession number: KJ716332). Meanwhile, four putatives sugars transporter genes, MGT04891 (accession number: XM_001482886), MGT05196 (accession number: XM_001482126), MGT05293 (accession number: XM_001482223), and MGT05860 (accession number: XM_001482047), were cloned from M. guilliermondii AS 2.1699, and the cloned sequences were confirmed to be the same as those in M. guilliermondii ATCC 6260 (Papon et al., 2013). Moreover, two transporters, Stp1p and AraEp, that we previously reported (Wang et al., 2013a; Zhang et al., 2013) were also chosen as candidates in the present work.

Twenty transporters, which were hypothesized to be either relatively efficient or 11

specific for D-xylose or L-arabinose transport in S.cerevisiae were selected and aligned with the eight candidates in the present work for phylogenetic diversity analysis. The results (Fig. 1) suggested that the eight transporters were related to different transport groups. Mgt05196p and Mgt05293p are close to Gxf1p, which is a relatively efficient heterologous D-xylose facilitator in S.cerevisiae (Runquist et al., 2010), while Stp1p and Mgt04891p are close to Gxs1p, which held higher preference ratio of xylose to glucose (Young et al., 2011; Young et al., 2014).

3.2. The monosaccharide transport capacities of the transporters

The sugar transport capacities of the collected eight transporters were compared in S. cerevisiae with the native transporter Gal2p, which has a better transport capacity among the 18 native hexose transporters to both D-xylose (Young et al., 2011) and L-arabinose (Subtil and Boles, 2011).

The hexose transport performance of the transporters was evaluated using an assay that tested whether they could endow the hxt null strain with the capacity to grow on the test hexoses (Fig. 2). Maltose was used as the reference carbon source because S. cerevisiae has specific transporters for this disaccharide, and therefore the hxt null strain can grow on this source (Farwick et al., 2014; Verho et al., 2011; Young et al., 2011; Young et al., 2014). The candidate transporters exhibited different hexose transport capacities. The native transporter Gal2p recovered the growth of the hxt null strain on all of the tested hexoses (Fig. 2, Line2) as previously reported (Farwick et al., 2014; Young et al., 2014). The heterologous proteins Mgt04891p, Mgt05196p, Mgt05293p, Mgt05860p, and Stp1p also recovered the strain growth on all of the tested hexoses. The strain expressing Tct2p displayed only very weak 12

growth on the hexoses, and the AraEp and Tct1p expressing strains did not grow on the hexoses (Fig. 2).

Natural S. cerevisiae cannot grow on D-xylose and L-arabinose, but can convert them into sugar alcohol (Matsushika et al., 2013; Peng et al., 2012; Wang et al., 2013b). Therefore, the pentose transport performance of the transporters was evaluated upon assaying the total intracellular accumulation of sugar and sugar alcohol (Du et al., 2010). The results (Fig. 3) indicated that little D-xylose or L-arabinose was detected in the control strain, which only expressed the empty plasmid. The heterologous transporters Mgt04891p, Mgt05196p, Mgt05293p, Mgt05860p, and Stp1p prominently improved the strain net D-xylose accumulation (Fig.3), as well as the Gal2p and heterologous reference Gxf1p, which promotes D-xylose transportation (Diao et al., 2013). These results suggested that these five proteins were transporters for both the pentoses and hexoses, since they also recovered the growth of the hxt null strain on the tested hexoses (Fig. 2). Conversely, the hxt null cells containing AraEp and Tct1p accumulated only a little bit D-xylose and L-arabinose than the control strain, respectively (Fig. 3), and they did not grow on the tested hexoses (Fig. 2). This result suggested that AraEp and Tct1p cannot transport hexoses, and they may transport D-xylose and L-arabinose but with low efficiency. The Tct2p, the other transporter cloned from T. cutaneum AS 2.571, only permit strain grow on hexoses weakly (Fig. 2) and accumulated both D-xylose and L-arabinose slightly (Fig. 3), suggesting that it is a low efficient transporter for pentoses and hexoses.

Constructing the D-xylose initial utilization pathway in the hxt null strain and assaying the contribution of the transporter to the growth of engineered strains on 13

D-xylose is another way to evaluate the D-xylose transport capacity (Young et al., 2011; Young et al., 2014). Therefore, xylose reductase (XR) and xylitol dehydrogenase (XDH) from S. stipitis, and xylulose kinase (XK) from S. cerevisiae were inserted into the rDNA locus of the specific transporter-expressing hxt null strain using an integration plasmid pYMIK-xy127 we previously reported (Wang et al., 2004). The growth of the resulting strains on D-xylose was tested. The control strain BSW4EYX only expressed XR-XDH-XK but not the transporter, thus it did not grow on D-xylose, as expected. The heterologous Mgt05196p provided the strain a growth capacity close to Gal2p (Fig. 4). The growth rates of strain expressing Gal2p, Mgt05196, and Mgt05293 were 0.019, 0.018, and 0.014 h-1, repectively, while other transporter expressing strain did permit obvious cell growth, including the, Mgt05860p (Fig. 4), even though it contributed to the highest D-xylose intracellular accumulation (Fig. 3A). 3.3. The type of D-xylose transporters, facilitator or H+ symporter

The studied D-xylose transporters in S. cerevisiae belong to two types, facilitator and symporter. Facilitators display a facilitated diffusion mechanism, and usually exhibit low uptake affinities but high efficiency. Symporters transport the substrate coupled with H + symport, and usually exhibit high affinities but sometimes block cell growth on D-xylose due to the energy-dependent transport process (Du et al., 2010; Leandro et al., 2006). H+ symporters cause significant pH increases due to the H+ transport across the membrane with the substrate (Du et al., 2010). The pH value changes (recorded as ΔpH) were determined (Fig. 5) in the presence of D-xylose. The pH of the Mgt05860p-expressing strain prominently increased, which was very different from the other strains. The results indicated that Mgt05860p might be an H+ 14

symporter, and thus may underlie the observation that Mgt05860p dramatically improved the intracellular accumulation of D-xylose in the hxt null strain (Fig. 3A), but did not permit cell growth when D-xylose was the sole carbon source (Fig. 4). The energy generated from the weak D-xylose metabolism was exhausted by D-xylose transport, and therefore this transporter could not support strain growth (Matsushika et al., 2013). Otherwise, another hypothesis is that most of the D-xylose molecules merely bind to the Mgt05860p and cannot enter cells for some reason, so that they were detected in the extract water in the intracellular accumulation assay, but did not afford the cell growth on D-xylose. In comparison, the other transporters did not influence the pH value (Fig. 5), suggesting that they were not symporters but facilitators (Du et al., 2010; Fonseca et al., 2007).

3.4. The characteristics of Mgt05196p for D-xylose transportation in hxt null S. cerevisiae

The fluorescence of the fusion protein Mgt05196p-GFP-expressing strain (see Figure 1 in Ref [Wang et al., 2015]) revealed that the heterologous transporter Mgt05196p was accurately targeted to the plasma membrane. The strains expressing Mgt05196p exhibited more D-xylose intracellular accumulation (Fig. 3A) and better growth capacity on D-xylose (Fig. 4) compared to the other heterologous transporters. Therefore, Mgt05196p was selected for further studies.

To study the contribution of the transporter Mgt05196p to D-xylose metabolism, the transporters were expressed in the hxt null strain BSW4EYX, which was derived from EBY.VW4000 (Wieczorke et al., 1999) and expresses D-xylose metabolic pathway genes. The control strain with no transporter but an empty plasmid did not 15

grow on either D-xylose or glucose. The strains with the transporters Mgt05196p or Gal2p grew on D-xylose (see Figure 2 in Ref [Wang et al., 2015]). During D-xylose fermentation, the maximum specific D-xylose consumption rate of the strains with Mgt05196p and Gal2p were almost equal with approximately 0.035 g h−1 g−1 DCW, which suggested that Mgt05196p and Gal2p might possess similar D-xylose transport capacities. In the fermentation of medium containing both D-xylose and glucose, the ratio of the specific consumption rate of D-xylose and glucose (X/G) represents the transport preference for these two sugars (Young et al., 2011). The X/G of the Mgt05196p- and Gal2p-containing cells was 0.09 and 0.06, respectively, indicating that Mgt05196p held a higher preference for D-xylose transport than Gal2p. Moreover, both of the Mgt05196p- and Gal2p- expressing strains displayed substrate-concentration-dependent specific growth rates (μ) on D-xylose, but not on glucose. Their specific growth rates on glucose were maintained at approximately 0.22 h-1, independent of the level of glucose that was presented (Fig. 6).

3.5. The crucial amino acid residues in Mgt05196p and their impact on D-xylose transport

According to the outward-facing and partly occluded conformation of E.coli D-xylose permease XylEp (Sun et al., 2012), a secondary structure of Mgt05196p (see Figure 3 in Ref [Wang et al., 2015]) was predicted using the online tool HMMTOP (http://www.sacs.ucsf.edu/cgi-bin/hmmtop.py; Tusnady and Simon, 2001), a 3D structure of Mgt05196p (see Figure 4 in Ref [Wang et al., 2015]) was modeled preliminarily using Discovery Studio software. Compared to XylEp, Mgt05196p contained 12 similar transmembrane helices, but longer intracellular sequences both at the N-terminus and C-terminus, which were ignored when the 3D model of 16

Mgt05196p was constructed (see Figure 3 in Ref [Wang et al., 2015]). The polar/aromatic amino acid residues within a distance of 5 Å to D-xylose in the model, and a conserved aromatic residue-rich motif in the transmembrane section 7 (TMS7) were selected. Additionally, some other amino acid residues were selected according to previous reports (Henderson and Baldwin, 2013; Iancu et al., 2013; Nijland et al., 2014; Sun et al., 2012). A total of 28 crucial amino acid residues in Mgt05196p were chosen and studied, including F69, D72, F79, K156, R164, Q199, I202, T203, Q284, E293, Q325, Q326, N330, N331, Y332, F333, F334, Y335, Y336, G337, N360, G383, I393, N411, K415, F432, V440, and W465 (more details in Ref [Wang et al., 2015]).

The total 28 predicted critical amino acid residues in Mgt05196p were first replaced with the simplest nonpolar residue alanine (Ala, A) following the previous report (Sun et al., 2012). These mutants contributed both to the intracellular D-xylose accumulation in the hxt null strain (see Figure 5 in Ref [Wang et al., 2015]) and the growth of the hxt null strain with the XR-XDH pathway on D-xylose or glucose (Table 2).The tendencies of the growth and intracellular D-xylose accumulation results roughly corresponded with each other. For convenience, when describing D-xylose accumulation, the background strain was EBY.VW4000, and when describing the growth on D-xylose or glucose, the background strain was BSW4EYX. Moreover, the mutants of the transporter Mgt05196p are represented by the mutation site directly, for example, Mgt05196p(D72A) is represented as D72A.

Expressing the mutants D72A, R164A, F333A, and Y336A allowed the strain to neither grow on D-xylose nor accumulate D-xylose. Expression of D72A, R164A, and Y336A did not enable the strain to grow on glucose. Replacing the residues F69, I202, Q326, N331, Y332, F334, and Y335 with Ala reduced the strain exponential growth 17

rates by 76%, 48%, 69%, 90%, 84%, 72%, and 79% on D-xylose, respectively, and reduced the net intracellular D-xylose accumulation by 22%, 54%, 55%, 88%, 67%, 64%, and 59% at the incubation time of 120 min, respectively. Moreover, the replacement of the residues F69, F333, and F334 with Ala reduced the exponential growth rates of the cells by 69%, 92%, and 52% on glucose, respectively. Additionally, compared to the Mgt05196p-expressing strain, the exponential growth rates of the strains expressing the mutants Q199A, N360A, and F432A increased by 26%, 24% and 34% on D-xylose, respectively, but their growth on glucose did not improve. The D-xylose net intracellular accumulation of the strain expressing the mutant F432A also increased by 14% at the incubation time of 120 min. The other chosen residues did not noticeably affect the transport of D-xylose and glucose.

Some of the residues of Mgt05196p were replaced with residues besides alanine. The mutant Q199K neither supported strain growth on sugars (Table 2) nor accumulated D-xylose (see Figure 5 in Ref [Wang et al., 2015]). The glucose and D-xylose transport activity of mutant N330M was lower than Mgt05196p, and the N331M mutant was even lower (Table 2). Mutation of the residue Y335 to F or W reduced the transport activity for D-xylose as well as Y335A (Table 2). Although the mutant Y336A did not transport D-xylose and glucose, the mutant Y336F supported the growth of strain on D-xylose and glucose (Table 2), and the net intracellular D-xylose accumulation was 85.9% of Mgt05196p (see Figure 5 in Ref [Wang et al., 2015]). The mutant Y336W also supported the growth of the strain on glucose and D-xylose, although the D-xylose growth capacity was very low (Table 2), and the net intracellular D-xylose accumulation was 50.5% of Mgt05196p (see Figure 5 in Ref [Wang et al., 2015]). These results suggested that the phenyl ring of Y336 might play 18

a crucial role in the D-xylose transport process. Mutation of the residue N360 to S, as well as to A (Table 2), had no influence on glucose transport (Table 2), but effectively increased the exponential growth rate of the strain by 32% on D-xylose, and also increased the net intracellular D-xylose accumulation of the strain by 10% at the incubation time of 120 min (Table 2 and Figure 5 in Ref [Wang et al., 2015]). Notably, the strain expressing the mutant N360F did not grow on glucose (Table 2), but did grow on D-xylose with an exponential growth rate equivalent to 91% of the Mgt05196p-expressing strain (Table 2).The net intracellular D-xylose accumulation at the incubation time of 120 min of the N360F-expressing strain was 92% of the Mgt05196p-expressing strain (see Figure 5 in Ref [Wang et al., 2015]). Additionally, the mutants N360Y and N360W also lost the glucose transport capacity (Table 2), but still maintained a modest transport capacity on D-xylose (Table 2 and Figure 5 in Ref [Wang et al., 2015]). These results indicate that the phenyl ring of the N360 residue might block the glucose transport process.

3.6. The characteristics of Mgt05196p (N360F) and its contribution on the xylose-utilizing strain

The mutant N360F transported D-xylose but not glucose, as mentioned above. The uptake capacity on other hexoses was also tested. Compared to growth on D-xylose, the strain expressing N360F showed no growth on glucose, fructose, galactose, or mannose (Fig. 7A), and therefore these hexoses are not substrates of N360F. During fermentation, the strain expressing Mgt05196p consumed nearly all of the 20 g L-1 glucose within 4 hours of high cell density fermentation, but the strain expressing N360F did not utilize glucose at all. Additionally, the strain expressing N360F utilized D-xylose normally, although the utilization capacity was reduced 19

when compared to the strain expressing Mgt05196p (Fig. 7B). In the fermentation in mixed glucose-xylose media, the N360F conferred strain growth on D-xylose without any inhibition by the presence of glucose, regardless of the glucose concentration (Fig. 7C). These results demonstrate that there is no glucose competitive inhibition on D-xylose transport for the transporter Mgt05196p (N360F).

4. Discussion In S. cerevisiae, efficient and specific pentose transport is one of prerequisites for metabolizing pentose and hexose efficiently and simultaneously. The eight heterologous sugar transporters from pentose utilizing strains were characterized in the present work. AraEp was reported to transport both L-arabinose and D-xylose in prokaryotic C. glutamicum (Kawaguchi et al., 2009; Sasaki et al., 2009); however, it only exhibited slight D-xylose transport capacity in eukaryotic S. cerevisiae, indicating that this bacterial transporter has an altered preference in S. cerevisiae. Mgt05196p demonstrated D-xylose transport capacity nearly equal to that of Gal2p (Fig.4, and Figure 2 in Ref [Wang et al., 2015]), which is the most efficient endogenous D-xylose transporter (Young et al., 2011). Moreover, both of Mgt05196p and Gal2p transporters exhibited concentration-dependent transport efficiency for D-xylose (Fig. 6). The maximum specific D-xylose consumption rate of the Mgt05196p-and Gal2p-overexpressing strains were also almost equal in the fermentation on D-xylose as the sole carbon source. The X/G preference ratios Mgt05196p were 50% higher than Gal2p in the competitive preference of D-xylose and glucose in the shake-flasks (see Figure 2 in Ref [Wang et al., 2015]), indicating that Mgt05196p had a higher D-xylose-to-glucose preference.

20

A total of 28 amino acid residues in Mgt05196p for sugar transport were studied. The crucial amino acid residues D72, R164, and Y336 for both glucose and D-xylose transport and F333 only for D-xylose were confirmed because the replacement of these residues with alanine (A) almost completely blocked the strain absorption of the relevant sugars (Table 2 and Figure 5 in Ref [Wang et al., 2015]). The replacement of amino acids F69, I202, Q326, N331, Y332, F334, and Y335 with A also reduced the exponential growth rates and the net intracellular D-xylose accumulation of the expressing strains (Table 2 and Figure 5 in Ref [Wang et al., 2015]), suggesting that these residues are also important for the transporter. The TMS7 was reported to be a discontinuous helix that facilitated a conformational change during sugar transport (Deng et al., 2014; Screpanti and Hunte, 2007). Our results demonstrated that change any amino acids of Y332 to Y336 to A obviously decrease the D-xylose transport, while only F333 and Y336 mutations affected the glucose transport much, suggesting that the aromatic residue enriched sequence YFFYY (from 332 to 336) in the conserved motif of the TMS7 of Mgt05196p has more influence on D-xylose transport than on glucose (Table 2 and Figure 5 in Ref [Wang et al., 2015]). As mentioned above, the mutant Y336A has no transport capacity; however, Y336F transported D-xylose and glucose well (Table 2 and Figure 5 in Ref [Wang et al., 2015]). This result indicated that the phenyl ring of Y336 has a greater effect than the hydroxyl group on transport function. The 3D structure model (see Figure 4 in Ref [Wang et al., 2015]) suggested that the residue Y336 is located outside of and vertical to the pore. The steric exclusion of the phenyl ring in the aromatic residues might contribute to control the effusion of D-xylose during the conformational change. The situation of the neighboring Y335 residue is quite different. Y335A and Y335F have similar glucose transport capacity with Mgt05196p, and similar decreased D-xylose 21

transport capacity. Thus, this neighboring residue does not function similarly to Y336.

The two conserved amino acids, D27 and R133, were predicted to build a salt bridge and be involved in the protonation of XylE in E.coli to control the open-outward or the inward-facing conformation (Henderson and Baldwin, 2013). These two amino acids are conserved in Gal2p and Mgt05196p. The replacement of these two sites, which are D72 and R164 in Mgt05196p, to nonpolar A disabled the transport of D-xylose and glucose (Table 2 and Figure 5 in Ref [Wang et al., 2015]) similar to XylE (Sun et al., 2012). These two conserved sites might be involved in the formation of a salt bridge in Mgt05196p to control the open position, although it is not necessary to couple H + symport for Mgt05196p transport capacity. Aside from D72 and R164, Q326, N331, Y335, and N360 were given the same characteristic alteration with the corresponding residues in XylE (Q289, N294, Y298, and N325), while the mutation of Q199, Q325, F432 and W465 were not. The mutation of Q199, Q325, and W465 had no obvious effect on transport capacity, while their corresponding residues in XylE (Q168, Q288, and W416) were demonstrated to be the crucial residues for D-xylose transportation. The mutation of F432 in the predicted TMS10 region of Mgt05196p had no impact on glucose transport, but increased D-xylose transport activity, while its corresponding site F383 in XylE is verified to be in direct contact with glucose. Moreover, changing the N330 in Mgt05196 to M did not affect the glucose transport but the xylose transport reduced by half, while changing the corresponding sites D340 in Hxt7p to M dramatically decreased its affinity to glucose (Kasahara and Kasahara, 2010). The T213 in Hxt7p were proven to be crucial residues for glucose transport, the longer side chain length led to higher affinity to glucose, the affinity of Hxt7p (T213A) to glucose was much lower than wild type 22

Hxt7p (Kasahara et al., 2011), while changing the corresponding sites T203 in Mgt05196p to A did not affect the glucose transport, only reduced the D-xylose transport. These results indicated that the conserved amino acids in homologous transporters might possess different functions.

The N360 residue in the TMS8 was verified to be important for D-xylose transport capacity in Mgt05196p in this study, which corresponded to the important residue N376 in Gal2p (Farwick et al., 2014) and N367 in fusion transporter Hxt36 (Nijland, et al., 2014).The mutant N360S effectively increased the D-xylose transport capacity. Moreover, the mutant N360F completely abolished the hexose transport capacity, but still maintained the D-xylose transport capacity without any inhibition by glucose. The cost of specificity, however, includes not only the loss of hexose transport capacity but also a part of the D-xylose transport capacity. The phenyl ring of the N360 residue might present a steric exclusion effect to block the glucose transport process. Our result is correspondence with Gal2p (N376F), which is xylose specific and glucose inhibit release but with a decreased transport capacity (Farwick et al., 2014) but different with the fusion transporter Hxt36 (Nijland, et al., 2014). Changing the N367 of Hxt36 to either G, A, V, L, M, I, S, T, C, F, or H can bring the glucose inhibit released xylose transport, and the N367A brought the most efficient xylose transport, which was >2 times of N367F. The difference of other residues in the active centre between Mgt05196, Gal2, and Hxt36 might responsible for this.

5. Conclusion In this study, the properties of eight sugar transporters were studied in S. cerevisiae. Among them, Mgt05196p from Meyerozyma guilliermondii was revealed 23

to be the most efficient D-xylose transporter. The study of point mutations suggested consistent or different functions of the conserved amino acids between the sugar transporters Mgt05196p and XylE, whose three dimensional structures were solved. D72, R164, and an aromatic residue enriched sequence YFFYY (332-336) were suggested to be the crucial residues for the D-xylose transport capacity of Mgt05196p. Furthermore, mutants were screened for efficient pentose utilization. The F432A and N360S mutants enhanced D-xylose transport activities. Notably, the N360F mutant specifically transported xylose without any glucose inhibition, highlighting its potential application in constructing glucose-xylose co-fermentation strains for biomass refining.

Acknowledgments This study was supported by the National High Technology Research and Development Program of China (2014AA021903, 2012AA022106), the National Key Technology R&D Program of China (2014BAD02B07), the National Natural Science Foundation of China (31270151, 31470166, and 31470163), the Project of National Energy Administration of China (NY20130402), the State Key Laboratory of Motor Vehicle Biofuel Technology (No. 2013004, KFKT2013002), and the Natural Science Foundation of Shandong Province (ZR2014CL003).

The authors thank Prof. Dr. Eckhard Boles from Institut für Molekulare Biowissenschaften Goethe-Universität Frankfurt for kindly providing the EBY.VW4000 strain.

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Figure legends

Fig. 1. Phylogenetic tree of the transporters. The cladogram of the cloned eight transporters and the functional pentose transporters was aligned using ClustalW and then calculated using the neighbor joining method with Mega 5. Sc, S. cerevisiae; Ci,C. intermedia;Ss, S. stipitis;Mg, M. guilliermondii;Tr, T. reesei;Dh, D. hansenii;Tc, T. cutaneum; At, A. thaliana;Ec, E. coli;Cg, C. glutamicum;Am, A. monospora;Nc, N. crassa

Fig. 2. The hexose transport capacity of the transporters. Four microliters of the 10-fold dilutions with an initial cells OD600 =1 were spotted on SD medium with 20 g L-1 of different sugars. The cells were cultured at 30 °C for 5 d. Strains: All of the strains were derived from the hxt null strain EBY.VW4000. The control was strain BSW4PP, which contained the empty plasmid. The other strains are represented by the transporters they expressed shown on the left side.

Fig. 3. The pentose transport capacity of the transporters. The seed cells were incubated in SD medium with either 20 g L-1 D-xylose (A) or 20 g L-1 L-arabinose (B) at 30°C for 30, 60 and 120 min, then the intracellular sugars were extract using ddH2O. The intracellular pentose was defined as the total amount of extracted pentose and pentose alcohol per gram in the dried cells (Du et al., 2010). The error bars represent the standard deviation of the biological triplicates. Compared to the control, significantly increased data (p