Strain and media
The Escherichia coli DH5∝ (Nippon gene, Japan) was used for all recombinant DNA manipulations. All the yeast strains used and constructed in this study are summarized in Additional file 2: Table S2. S. cerevisiae strains MT8-1  and W303-1B  were used as the host strains for the gene expression and fermentation, respectively. E. coli was grown in Luria–Bertani medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l sodium chloride) containing 100 mg/l ampicillin or 50 mg/l kanamycin. S. cerevisiae strains were aerobically cultivated at 30 °C in synthetic medium [SD medium; 20 g/l glucose and 6.7 g/l yeast nitrogen base without amino acids (Difco/BD Diagnostic Systems, MD, USA) with appropriate supplements.
Adaptation to xylose is known to greatly improve the xylose fermentation abilities of the recombinant yeast strains [29, 35]. Thus, to exclude the effect of adaptation, we used glucose as the carbon source to prepare the pre-cultures used for the enzyme and growth assays, and also in fermentation experiments.
PCR amplification and cloning procedure
PCR reactions were carried out either with PrimeSTAR HS DNA polymerase or with Ex Taq HS DNA polymerase (Takara Bio Inc., Japan). The PCR primers used in this study are listed in Additional file 10: Table S4. PCR products were cloned into pCR2.1-TOPO using the TOPO TA cloning kit or pCR-BluntII-TOPO using the ZERO Blunt TOPO PCR cloning kit (Thermo Fisher Scientific Inc., MA, USA). For generating plasmids, the DNA fragments were cloned into appropriate vectors using the In-Fusion Advantage PCR Cloning kit (Takara Bio Inc.).
Isolation of novel XI genes from cDNA library
A cDNA library, previously prepared from the protists residing in the R. speratus hindgut , was used in this study for the cloning of novel XI genes. In order to obtain a xylose isomerase gene from this cDNA library, degenerate primers mXI-F1 and mXI-R1, which were designed based on the alignment of DNA sequences of known XI genes obtained from GenBank, were synthesized. Using these degenerate primers, an approximately 400-bp DNA fragment was amplified from this cDNA library. The purified fragment was cloned and sequenced. Consequently, various plasmids containing inserts exhibiting partial sequence similarities to known XI genes were obtained. The 5′- and 3′-regions of these sequences were amplified from the cDNA library by PCR using appropriate combinations of the following primer pairs: RsA-F, RsB-R, RsC1-R, RsC2-F, RsD-R, RsE-F, and RsF-R, which were designed from the revealed partial sequences of inserts, and Lib-F and Lib-R, which were designed from the vector sequence of the 5′- and 3′-flanking regions of the cDNA insert. Amplified DNA fragments were cloned and sequenced. Next, full-length DNA fragments were obtained by PCR using the cDNA library as the template and appropriate primer pairs (RsA-termR, RsB-termF, RsC1-termF, RsC2-termR, RsD-termF, RsE-termR, and RsF-termF, all of which were designed from the 5′- or 3′-regions of the gene sequence, and Lib-F and Lib-R). Amplified DNA fragments were cloned and sequenced. Finally, the entire cDNA sequence of each clone was amplified by PCR from the cDNA library using one of the following appropriate primer pairs: RsXI-A-IF-F/RsXI-A-IF-R, RsXI-B-IF-F/RsXI-B-IF-R, RsXI-C1-IF-F/RsXI-C1-IF-R, RsXI-C2-IF-F/RsXI-C2-IF-R, RsXI-D-IF-F/RsXI-D-IF-R, RsXI-E-IF-F/RsXI-E-IF-R, or RsXI-F-IF-F/RsXI-F-IF-R. Each amplified fragment was then cloned into the SacII/XhoI site of pRS436GAP (DDBJ Accession Number: AB3048629), and complete nucleotide sequence of each amplified DNA fragment was obtained. A general map of the resulting plasmid, created as above by inserting a RsXI gene into pRS436GAP, is shown in Additional file 11: Figure S7a. All the generated plasmids are listed in Additional file 7: Table S3.
The amino acid sequences derived from the full-length cDNA clones and DNA sequences of previously reported XIs were aligned and the phylogenetic tree was plotted using the genetic information analysis software GENETYX version 10 (GENETYX, Japan). The phylogenetic tree was constructed with 1000 boot strap replicates using the neighbor-joining method .
To determine the origin of RsXI-C1, we fractionated symbiotic protists and bacteria, and performed RT-PCR on cDNA prepared from each fraction.
Excised hindguts from 30 individuals of R. speratus were placed into a 1.5-ml microfuge tube along with 100 μl of solution U  and disrupted by plastic pestle on ice. The resulting suspension was then filtered through a 100-μm pore size nylon mesh to remove the gut debris. The filtrate was centrifuged at 500 rpm for 5 min using a swing bucket rotor. The supernatant was used as the bacterial fraction and the pellet was used as the protistan fraction. To clean up the bacterial fraction, the supernatant was collected after centrifugation at 500 rpm for 5 min, and this cycle was repeated 4 times. The resulting supernatant was subsequently centrifuged at 15,000 rpm for 1 min and the resulting bacterial pellet was saved and the supernatant was discarded. To clean up the protistan fraction, the protistan pellet (see above) was resuspended in 100 μl of solution U, centrifuged at 500 rpm for 5 min, and the supernatant was discarded after centrifugation. This cycle was repeated 4 times. All centrifugation steps were performed at 4 °C.
Total RNA was purified from each fraction by RNeasy mini kit (QIAGEN, Hilder, Germany). RNA was eluted from the spin column with 30 μl of RNase-free water included in the kit. Eluted RNA was used as a template for RT-PCR.
The purified total RNA was reverse transcribed with the degenerated complementary primer RsXI_C1_Cend_comp, which was designed based on the C-terminus amino acid sequence of RsXI-C1. The transcribed mixture was then used in a PCR amplification reaction using the degenerate primer RsXI_C1_Cend_comp and the degenerated primer RsXI_C1_Nend, which was designed based on the N-terminus amino acid sequence of RsXI-C1.
Reverse transcription reaction was performed at 50 °C for 10 min using 200 units of Superscript IV (Thermo Fisher Scientific Inc.) in a total volume of 20 μl that contained 2 pmol of each primer, 1× Superscript IV buffer, 0.5 mM dNTP, and 5 mM DTT, following which the reverse transcriptase was inactivated by incubating the reaction mixture at 80 °C for 10 min. After the reaction, the remaining RNA was removed by treatment with RNase H (2 units per reaction) at 37 °C for 30 min.
The reaction product, obtained as above, was used for the PCR amplification. PCR reaction was carried out in 50 μl total volume and each reaction mixture contained 1× ExTaq buffer (Takara Bio Inc.), 50 pmol of each primer, 0.2 mM dNTP, 5 units ExTaq (Takara Bio Inc.), and 5 μl of transcribe product. The PCR reaction cycle used for amplification was as follows: 94 °C for 30 s, 50 °C for 45 s, and 72 °C for 2 min (total 30 cycles). The obtained amplicon was analyzed by 1% agarose electrophoresis.
In situ hybridization
In situ hybridization was performed as described previously  with some modification. Excised hindguts isolated from 10 termites were placed into a 1.5-ml microfuge tube along with 100 μl of solution U and disrupted by plastic pestle on ice. The resulting suspension was then filtered through a 100-μm pore size nylon mesh to remove the gut debris.
Symbiotic protists were collected from the resulting suspension by centrifugation at 500 rpm for 3 min at 4 °C and the pellet was washed 3-times with 100 μl of solution U. Washed protists were fixed by incubating with solution U containing 4% formaldehyde at room temperature for 15 min. After fixation, suspended protists were dehydrated by sequentially washing with 50% ethanol for 5 min, 80% ethanol for 5 min, and finally with 100% ethanol for 5 min. After dehydration, suspended protists were fixed on glass slides by spotting and drying.
Spotted specimen was treated with pre-hybridization solution (2 pmol/μl RsXI_C1_Nend, 300 mM NaCl, 30 mM sodium citrate, and 0.01% SDS) at 37 °C for 30 min. After the removal of pre-hybridization solution, hybridization was carried out in hybridization solution that contained 2 pmol/μl fluorescein-labeled C_nested_comp (anti-sense) probe or fluorescent-labeled C_nested (sense) probe, 300 mM NaCl, 30 mM sodium citrate, and 0.01% SDS, and slides were incubated with this solution at 37 °C for 1 h. 5′ end fluorescein-labeled oligonucleotide probes were synthesized by Eurofins genomics K.K. (Tokyo, Japan). Probe sequences are listed in Additional file 10: Table S4.
After hybridization, slides were washed twice with PBS-T (0.4% NaCl, 0.01% KCl, 0.145% Na2HPO4·12H2O, 0.01% KH2PO4, 0.2% Tween 20) at 37 °C for 15 min to remove excess probes. Slides were then sealed with Vector Shield (Vector Laboratories, CA, USA) and observed under a fluorescent microscope (Model IX70, Olympus, Japan).
Construction of plasmids
Expression plasmids were constructed to overexpress the following genes of the non-oxidative pentose phosphate pathway: XKS1 encoding xylulokinase, RK11 encoding ribulose 5-phosphate isomerase, RPE1 encoding ribulose 5-phosphate epimerase, TKL1 encoding transketolase, and TAL1 encoding transaldolase. The integration vector pXhisHph-HOR7p-ScXK (Additional file 11: Figure S7b), which is targeted to the HIS3 loci in chromosome XV, was used for the construction of XKS1 overexpression plasmid. The integration vector pXAd3H-HOR7p-ScTAL1-ScTKL1 (Additional file 11: Figure S7c), which is targeted to the upstream region of ADH3 in chromosome XIII, was used for the construction of TAL1 and TKL1 overexpression plasmids. The integration vector pXGr3L-HOR7p-ScRPE1-ScRKI1 (Additional file 11: Figure S7d), which is targeted to the GRE3 loci in chromosome VIII, was used for the construction of RPE1 and RKI1 overexpression plasmids. All the genes were expressed under the control of the HOR7 promoter and the CYC1 terminator.
A codon-optimized RsXI-C1 (RsXI-C1O) was synthesized based on the amino acid sequence of RsXI-C1. The XI gene from the Piromyces sp. E2 (PiXI) was synthesized based on the DNA sequence acquired from the GenBank (GenBank Accession Number: AJ249909.1). The codon-optimized version of PiXI (PiXIO) and codon-optimized XI gene from C. phytofermentans DSM18823 (CpXIO) were synthesized based on the amino acid sequences acquired from the GenBank (GenBank Accession Numbers: CAB76571.1 and ABX41597.1, respectively). These synthesized DNA fragments, which were custom synthesized from GenScript (NJ, USA), were individually cloned into the SacII/XhoI site of pRS436GAP, and the resulting plasmids were designated as pRS436GAP-PiXI, pRS436GAP-PiXIO, pRS436GAP-RsXIC1O, and pRS436GAP-CpXIO, respectively (Additional file 7: Table S3).
Yeasts were transformed with a Frozen-EZ Yeast transformation II kit (Zymo Research, CA, USA). About 1 μg of episomal plasmid or about 5 μg of linearized integration vector was used for the transformation, followed by selection on selective SD agar plates. Resulting yeast strains are summarized in Additional file 2: Table S2.
To create a strain that would overexpress PPP-related genes, plasmids pXhisHph-HOR7p-ScXK, pXAd3H-HOR7p-ScTAL1-ScTKL1, and pXGr3L-HOR7p-ScRPE1-ScRKI1, each digested with restriction enzyme Sse8387I, were introduced into the strain MT8-1 and the resulting strain was named PP600.
W303-1B-based PPP overexpression strain was constructed as follows. First, the ADE2 (GenBank Accession Number: M59824) was amplified by PCR from the genomic DNA purified from S. cerevisiae S288C and using the primer pair ADE2 + 1F and ADE2 + 1716R. The resulting amplification product was used to transform the W303-1B strain that complemented the ade2 mutation. The resulting strain, which was named W303-1BA, was transformed with linearized DNA fragments obtained from the Sse8387I digested plasmids pXhisHph-HOR7p-ScXK, pXAd3H-HOR7p-ScTAL1-ScTKL1, and pXGr3L-HOR7p-ScRPE1-ScRKI1. The resulting strain was named W600.
Finally, the TRP1 (GenBank Accession Number: 851570) and its neighboring region were amplified by PCR from the genomic DNA purified from S. cerevisiae S288C and using the primer pair TRP1M-F and TRP1M-R. The resulting amplification product was used to transform W600 strain to complement the trp1 mutation, and the resulting strain was named W600W. Strains PP600 and W600W were transformed with the XI gene expression plasmids. Descriptions of the strains created in this study are listed in Additional file 2: Table S2.
Yeast transformants were cultivated at 30 °C for 24 h in SD medium, following which cells were harvested by centrifugation, washed twice with sterile distilled water, and then washed twice with 100 mM phosphate buffer (pH 7.0). Washed cell pellets were disrupted with glass beads (diameter, 0.3 mm, Yasui Kikai, Japan) using Micro mixer E-36 (Taitec Corporation, Japan) or Multi-beads shocker (Yasui Kikai, Japan). Cell extracts were centrifuged at 12,000 rpm at 4 °C for 5 min, and the resulting supernatants were collected as crude cell extracts. The total protein concentration in the crude cell extract was determined using the Quick Start protein assay kit (Bio-Rad, CA, USA).
XI activities in the crude cell extracts of recombinant yeast strains were determined by two different assay methods. One method, which has been modified from a previously described method , was used for measuring the XI activity of novel XI gene products. In brief, the assay was carried out in a reaction mixture that contained 50 mM maleic acid (pH 6.85), 10 mM MgSO4, 1 mM CoCl2, 1 mM MnCl2, and 10 mM xylose. The reaction was initiated by the addition of crude cell extract, and then the reaction mixture was incubated at 30 °C for 30 min. After the incubation period was over, cysteine-carbazole-sulfuric acid regent, containing 2.7 mM cysteine, 0.22 mM carbazole, and 66% H2SO4, was added to the reaction mixture. The mixture was incubated for 20 min at room temperature, following which its absorbance at 540 nm was measured to determine the amount of d-xylulose produced using a d-xylulose standard curve. One unit (U) of enzyme activity was defined as the activity that produced one μmol d-xylulose per min at 30 °C.
The other method was used to determine the kinetic properties of XIs as described previously . In this method, the XI activity was determined spectrophotometrically by measuring the oxidation of NADH at 340 nm. The assay mixture contained 100 mM Tris–HCl (pH 7.5), 10 mM MgSO4, 0.15 mM NADH, 2 U sorbitol dehydrogenase (Wako, Japan), and crude cell extract. The reaction was performed at 30 °C, and it was initiated by the addition of d-xylose to a final concentration of 5–250 mM.
Yeast strains were grown on SD medium at 30 °C for 24 h. After cultivation, cells were collected, washed with sterile distilled water, and then inoculated into SX medium that contained 6.7 g/l of yeast nitrogen base without amino acids and 20 g/l of xylose as the sole carbon source. The initial cell density was adjusted to an optical density at 600 nm (OD600) of 0.05. For the growth assay, cells were grown in L-shaped test tubes at 30 °C with shaking (70 rpm) in a Bio-photorecorder (model TVS062CA, ADVANTEC, Japan).
Anaerobic batch fermentation
Anaerobic batch fermentations were carried out in 100-ml medium bottles sealed with caps equipped with gas check valves. Yeast strains were aerobically pre-cultivated in SD medium for 3 days at 30 °C. Each pre-culture was separately inoculated into SD medium and aerobically cultivated for 24 h at 30 °C. Cells were collected and washed with distilled water. For xylose fermentation assay, these strains were inoculated into 50 ml fermentation medium, which contained 6.7 g/l of yeast nitrogen base without amino acids and 50 g/l of xylose. For glucose/xylose fermentation assay, these strains were inoculated into 50 ml fermentation medium, which contained 6.7 g/l of yeast nitrogen base without amino acids, 30 g/l of glucose, and 20 g/l of xylose. The initial cell density in the fermentation medium was adjusted to OD600 of 10. All fermentation experiments were performed at 30 °C with agitation (100 rpm).
Concentrations of glucose, xylose, glycerol, xylitol, and ethanol in the fermentation medium were analyzed by high-performance liquid chromatography (HPLC) (Prominence, Shimadzu, Japan) on a HPX-87H column (Bio-Rad), used together with a refractive index detector (model RID-10A, Shimadzu). The HPLC system was operated at 60 °C using 0.05% H2SO4 as the mobile phase (flow rate, 0.6 ml/min).
Microaerobic fermentations were carried out in 96-well Storage Blocks (Corning Incorporated, NY, USA). Yeast strains were aerobically pre-cultured in 96-well Storage Blocks (1 ml of SD medium per well), sealed with sterile breathable Nunc Sealing Tape (Thermo Fisher Scientific Inc.), with shaking at 1500 rpm in an M-BR-022UP constant temperature incubator shaker (Taitec Corporation) for 24 h at 30 °C. Next, 200 μl of the pre-culture was used to inoculate 1 ml of SD medium and cultured under the same condition for 24 h. Cells were harvested, washed twice with 1 ml of sterile water, and then suspended in 0.2 ml of sterile water to prepare yeast suspensions. Fermentation test was performed under the following conditions. One milliliter of SX medium, containing 20 g/l of xylose as the sole carbon source, was placed into each well of a 96-well storage block, to which yeast suspension was added to a final OD600 of 1.0 (for comparing xylose fermentation by the mutants generated by saturated mutagenesis) or to a final OD600 of 10.0 (for comparing xylose fermentation by other mutated XIs). For establishing a microaerobic condition, each well was hermetically sealed with Titer Stick HC film (Kajixx Co., Ltd., Japan), and fermentation was carried out in the M-BR-022UP incubator at 30 °C with shaking at 1500 rpm. An aliquot of the fermented liquid was taken out at different times as indicated and analyzed by HPLC.
Introduction of mutation in RsXI-C1 and growth-based screening of mutants
To introduce random mutations in the XI genes, an error-prone PCR was carried out with GeneMorph II kit (Agilent technologies, CA, USA) using pRS436GAP-RsXIC1O as the template and primer pair pRSSacII-AAA-ATG-F4 and pRSXhoI-TAA-R3; sequences of these primers are listed in Additional file 10: Table S4. Using this method, we were able to introduce on average 3 mutations per 1000 bases in the DNA fragment (error rate 0.3%) by error-prone PCR.
A yeast centromeric vector pRS316GAP was constructed for introducing the mutated XI genes into yeast. The P
region was amplified from the plasmid pRS436GAP by PCR using the primers TDH3p-CYC1t-IF-F and TDH3p-CYC1t-IF-R (Additional file 10: Table S4). The amplified DNA fragment was inserted into the PvuII-digested pRS316 (NBRP Accession Number: BYP562) to generate pRS316GAP (Additional file 11: Figure S7e). In this plasmid, the cloned genes are under the control of the TDH3 promoter and CYC1 terminator.
The mutated RsXI-C1O library, produced by error-prone PCR (see above), was mixed with SacII and XhoI digested pRS316GAP, and directly introduced into the W600W strain using the gap repair cloning protocol . The resulting yeast transformants were cultured in SD medium for 2 days. The non-mutated DNA fragment (control), amplified by PCR using pRS436GAP-RsXIC1O as the template, was introduced into the W600W strain in the same way as described above.
One hundred microliter of the mutant library culture was added to 5 ml of SX medium, and incubated at 30 °C with shaking at 70 rpm in the Bio-photorecorder. After 7 days of cultivation, cells were harvested from the culture medium, and resuspended in 5 ml of fresh SX medium to a final OD600 of 0.1. After culturing for 7 days in the Bio-photorecorder, cells were recovered from the culture medium and then spread on an SX agar plate and incubated at 30 °C. Colonies growing faster than the control were selected and cultured on an SD agar plate. Twenty clones were selected for the growth experiment in SX medium, from which top 10 strains exhibiting better growth rates than the control strain were chosen for plasmid extraction. Plasmids were extracted using the Zymoprep Yeast Plasmid Minipreparation kit (Zymo research). DNA inserts of these extracted plasmids were sequenced completely to identify the introduced mutation. These plasmids were subsequently used to retransform W600W.
Construction of the saturation mutagenesis library
An amino acid point mutation library, targeting the asparagine 337 (N337) residue of RsXI, was constructed by site-directed mutagenesis using the plasmid pRS316GAP-RsXIC1O as the template, primers listed in Additional file 10: Table S4, and a Quick Change Lightning MultiSite-Directed Mutagenesis kit (Agilent Technologies) following the protocol provided with the kit. Consequently, 18 plasmids, each expressing a different single amino acid substitution mutant (except asparagine and threonine), were obtained (Additional file 7: Table S3). Eighteen different strains were generated by transforming the W600W strain separately with each mutant plasmid. Resulting strains are listed in Additional file 2: Table S2.
Introduction of mutation into other XIs
Preparation of DNA templates for introducing a point mutation was carried out as follows. The coding regions of PiXIO and CpXIO were amplified from the plasmids pRS436GAP-PiXIO and pRS436GAP-CpXIO, respectively, by PCR. The resulting DNA fragments were inserted into the SacII/XhoI digested pRS316GAP plasmid to construct pRS316GAP-PiXIO and pRS316GAP-CpXIO, respectively. A synthetic LlXI (LlXIO), codons optimized for expression in yeast, was custom synthesized (GenScript Corporation); the amino acid sequence of LlXI was acquired from the GenBank (RefSeq Accession Number: WP_057720788.1). The resulting DNA fragment was cloned into the SacII/XhoI digested pRS316GAP to construct pRS316GAP-LlXIO.
Plasmids pRS316GAP-PiXIO, pRS316GAP-CpXIO, and pRS316GAP-LlXIO were used as templates for the PCR-based site-directed mutagenesis. For each XI gene, four different single amino acid substitution mutants were created; thus, the respective asparagine residue in each XI was substituted with cysteine, threonine, valine, and alanine. Corresponding plasmids harboring these substitution mutants are listed in Additional file 7: Table S3. The resulting plasmids were individually introduced into the W600W strain to create the XI mutant expressing strains (Additional file 2: Table S2).
Protein structure prediction
The three-dimensional predicted model of RsXI-C1 and N337C mutant of RsXI-C1 were generated based on the crystal structure of Bacteroides thetaiotaomicron xylose isomerase (Protein Data Bank code 4XKM)  using Discovery Studio (Dassault Systèmes, France).